Recombinant Anti-VLA4 Antibody Molecules

ABSTRACT

The present invention disclosed recombinant anti-VLA-4 antibody molecules, including humanized recombinant anti-VLA-4 antibody molecules. These antibodies are useful in the treatment of specific and non-specific inflammation, including asthma and inflammatory bowel disease. In addition, the humanized recombinant anti-VLA-4 antibodies disclosed can be useful in methods of diagnosing and localizing sites of inflammation.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/606,388, filed Nov. 29, 2006, which is acontinuation of U.S. patent application Ser. No. 10/428,662, filed onMay 2, 2003, now U.S. Pat. No. 7,157,086, which is a continuationapplication of U.S. patent application Ser. No. 08/454,899, filed on May31, 1995, now U.S. Pat. No. 6,602,503, which is a continuation-in-partapplication of U.S. patent application Ser. No. 08/004,798, filed onJan. 12, 1993, now abandoned, and is a continuation-in-part applicationof International Patent Application Serial No. PCT/US94/00266, filed onJan. 7, 1994. The contents of the prior applications are herebyincorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to recombinant anti-VLA-4 antibodymolecules, including humanized recombinant anti-VLA-4 antibodymolecules.

BACKGROUND OF THE INVENTION A. Immunoglobulins and Monoclonal Antibodies

Natural immunoglobulins have been known for many years, as have thevarious fragments thereof, such as the Fab, (Fab′)₂ and Fc fragments,which can be derived by enzymatic cleavage. Natural immunoglobulinscomprise generally a Y-shaped molecule having an antigen-binding sitetowards the free end of each upper arm. The remainder of the structure,and particularly the stem of the Y, mediates the effector functionsassociated with immunoglobulins.

Specifically, immunoglobulin molecules are comprised of two heavy (H)and two light (L) polypeptide chains, held together by disulfide bonds.Each chain of an immunoglobulin chain is divided into regions ordomains, each being approximately 110 amino acids. The light chain hastwo such domains while the heavy chain has four domains. The amino acidsequence of the amino-terminal domain of each polypeptide chain ishighly variable (V region), while the sequences of the remaining domainsare conserved or constant (C regions). A light chain is thereforecomposed of one variable (V_(L)) and one constant domain (C_(L)) while aheavy chain contains one variable (V_(H)) and three constant domains(CH₁, CH₂ and CH₃). An arm of the Y-shaped molecule consists of a lightchain (V+C_(L)) and the variable domain (V_(H)) and one constant domain(CH₁) of a heavy chain. The tail of the Y is composed of the remainingheavy chain constant domains (CH₂+CH₃). The C-terminal ends of the heavychains associate to form the Fc portion. Within each variable region arethree hypervariable regions. These hypervariable regions are alsodescribed as the complementarity determining regions (CDRs) because oftheir importance in binding of antigen. The four more conserved regionsof the variable domains are described as the framework regions (FRs).Each domain of an immunoglobulin consists of two beta-sheets heldtogether by a disulfide bridge, with their hydrophobic faces packedtogether. The individual beta strands are linked together by loops. Theoverall appearance can be described as a beta barrel having loops at theends. The CDRs form the loops at one end of the beta barrel of thevariable region.

Natural immunoglobulins have been used in assay, diagnosis and, to amore limited extent, therapy. However, such uses, especially in therapy,have been hindered by the polyclonal nature of natural immunoglobulins.A significant step towards the realization of the potential ofimmunoglobulins as therapeutic agents was the discovery of techniquesfor the preparation of monoclonal antibodies (MAbs) of definedspecificity, Kohler et al., 1975 [1]. However, most MAbs are produced byfusions of rodent (i.e., mouse, rat) spleen cells with rodent myelomacells. They are therefore essentially rodent proteins.

By 1990, over 100 murine monoclonal antibodies were in clinical trials,particularly in the U.S. and especially for application in the treatmentof cancer. However, by this time it was recognized that rejection ofmurine monoclonal antibodies by the undesirable immune response inhumans termed the HAMA (Human Anti-Mouse Antibody) response was a severelimitation, especially for the treatment of chronic disease. Therefore,the use of rodent MAbs as therapeutic agents in humans is inherentlylimited by the fact that the human subject will mount an immunologicalresponse to the MAb and either remove the MAb entirely or at leastreduce its effectiveness. In practice MAbs of rodent origin may not beused in a patient for more than one or a few treatments as a HAMAresponse soon develops rendering the MAb ineffective as well as givingrise to undesirable reactions. In fact, a HAMA response has beenobserved in the majority of patients following a single injection ofmouse antibody, (Schroff et al., 1985 [2]). A solution to the problem ofHAMA is to administer immunologically compatible human monoclonalantibodies. However, the technology for development of human monoclonalantibodies has lagged well behind that of murine antibodies (Borrebaecket al., 1990 [3]) such that very few human antibodies have proved usefulfor clinical study.

Proposals have therefore been made for making non-human MAbs lessantigenic in humans. Such techniques can be generically termed“humanization” techniques. These techniques generally involve the use ofrecombinant DNA technology to manipulate DNA sequences encoding thepolypeptide chains of the antibody molecule. The use of recombinant DNAtechnology to clone antibody genes has provided an alternative whereby amurine monoclonal antibody can be converted to a predominantlyhuman-form (i.e., humanized) with the same antigen binding properties(Riechmann et al., 1988 [4]). Generally, the goal of the humanizingtechnology is to develop humanized antibodies with very little orvirtually no murine component apart from the CDRs (see, e.g., Tempest etal., 1991 [5]) so as to reduce or eliminate their immunogenicity inhumans.

Early methods for humanizing MAbs involved production of chimericantibodies in which an antigen binding site comprising the completevariable domains of one antibody is linked to constant domains derivedfrom another antibody. Methods for carrying out such chimerizationprocedures have been described, for example, in EP 120694 [6], EP 125023[7], and WO 86/01533 [8]. Generally disclosed are processes forpreparing antibody molecules having the variable domains from anon-human MAb such as a mouse MAb and the constant domains from a humanimmunoglobulin. Such chimeric antibodies are not truly humanized becausethey still contain a significant proportion of non-human amino acidsequence, i.e., the complete non-human variable domains, and thus maystill elicit some HAMA response, particularly if administered over aprolonged period, Begent et al., 1990 [9]. In addition, it is believedthat these methods in some cases (e.g., EP 120694 [6]; EP 125023 [7] andU.S. Pat. No. 4,816,567 [10]) did not lead to the expression of anysignificant quantities of Ig polypeptide chains, nor the production ofIg activity without in vitro solubilization and chain reconstitution,nor to the secretion and assembly of the chains into the desiredchimeric recombinant antibodies. These same problems may be noted forthe initial production of non-chimeric recombinant antibodies (e.g.,U.S. Pat. No. 4,816,397 [11]).

B. Humanized Recombinant Antibodies and CDR-Grafting Technology

Following the early methods for the preparation of chimeric antibodies,a new approach was described in EP 0239400 [12] whereby antibodies arealtered by substitution of their complementarity determining regions(CDRs) for one species with those from another. This process may beused, for example, to substitute the CDRs from human heavy and lightchain Ig variable region domains with alternative CDRs from murinevariable region domains. These altered Ig variable regions maysubsequently be combined with human Ig constant regions to createdantibodies which are totally human in composition except for thesubstituted murine CDRs. Such murine CDR-substituted antibodies would bepredicted to be less likely to elicit a considerably reduced immuneresponse in humans compared to chimeric antibodies because they containconsiderably less murine components.

The process for humanizing monoclonal antibodies via CDR grafting hasbeen termed “reshaping”. (Riechmann et al., 1988 [4]; Verhoeyen et al.,1988 [13]). Typically, complementarity determining regions (CDRs) of amurine antibody are transplanted onto the corresponding regions in ahuman antibody, since it is the CDRs (three in antibody heavy chains,three in light chains) that are the regions of the mouse antibody whichbind to a specific antigen. Transplantation of CDRs is achieved bygenetic engineering whereby CDR DNA sequences are determined by cloningof murine heavy and light chain variable (V) region gene segments, andare then transferred to corresponding human V regions by site-directedmutagenesis. In the final stage of the process, human constant regiongene segments of the desired isotype (usually gamma 1 for C_(H) andkappa for C_(L)) are added and the humanized heavy and light chain genesare coexpressed in mammalian cells to produce soluble humanizedantibody.

The transfer of these CDRs to a human antibody confers on this antibodythe antigen binding properties of the original murine antibody. The sixCDRs in the murine antibody are mounted structurally on a V region“framework” region. The reason that CDR-grafting is successful is thatframework regions between mouse and human antibodies may have verysimilar 3-D structures with similar points of attachment for CDRs, suchthat CDRs can be interchanged. Nonetheless, certain amino acids withinframework regions are thought to interact with CDRs and to influenceoverall antigen binding affinity. The direct transfer of CDRs from amurine antibody to produce a recombinant humanized antibody without anymodifications of the human V region frameworks often results in apartial or complete loss of binding affinity.

In Riechmann et al., 1988 [4] and WO 89/07454 [14], it was found thattransfer of the CDR regions alone (as defined by Kabat et al., 1991 [15]and Wu et al., 1970 [16]) was not sufficient to provide satisfactoryantigen binding activity in the CDR-grafted product. Riechmann et al.1988 [4] found that it was necessary to convert a serine residue atposition 27 of the human sequence to the corresponding rat phenylalanineresidue to obtain a CDR-grafted product having satisfactory antigenbinding activity. This residue at position 27 of the heavy chain iswithin the structural loop adjacent to CDR1. A further construct whichadditionally contained a human serine to rat tyrosine change at position30 of the heavy chain did not have a significantly altered bindingactivity over the humanized antibody with the serine to phenylalaninechange at position 27 alone. These results indicate that changes toresidues of the human sequence outside the CDR regions, for example, inthe loop adjacent to CDR1, may be necessary to obtain effective antigenbinding activity for CDR-grafted antibodies which recognize more complexantigens. Even so, the binding affinity of the best CDR-graftedantibodies obtained was still significantly less than the original MAb.

More recently, Queen et al., 1989 [17] and WO 90/07861 [18] havedescribed the preparation of a humanized antibody that binds to theinterleukin 2 receptor, by combining the CDRs of a murine MAb (anti-Tac)with human immunoglobulin framework and constant regions. They havedemonstrated one solution to the problem of the loss of binding affinitythat often results from direct CDR transfer without any modifications ofthe human V region framework residues; their solution involves two keysteps. First, the human V framework regions are chosen by computeranalysts for optimal protein sequence homology to the V region frameworkof the original murine antibody, in this case, the anti-Tac MAb. In thesecond step, the tertiary structure of the murine V region is modelledby computer in order to visualize framework amino acid residues whichare likely to interact with the murine CDRs and these murine amino acidresidues are then superimposed on the homologous human framework. Theirapproach of employing homologous human frameworks with putative murinecontact residues resulted in humanized antibodies with similar bindingaffinities to the original murine antibody with respect to antibodiesspecific for the interleukin 2 receptor (Queen et al., 1989 [17]) andalso for antibodies specific for herpes simplex virus (HSV) (Co. et al.,1991 [19]). However, the reintroduction of murine residues into humanframeworks (at least 9 for anti-interleukin 2 receptor antibodies, atleast 9 and 7 for each of two anti-HSV antibodies) may increase theprospect of HAMA response to the framework region in the humanizedantibody. Bruggemann et al., 1989 [20] have demonstrated that human Vregion frameworks are recognized as foreign in mouse, and so,conversely, murine modified human frameworks might give rise to animmune reaction in humans.

According to the above described two step approach in WO 90/07861 [18],Queen et al. outlined four criteria for designing humanizedimmunoglobulins. The first criterion is to use as the human acceptor theframework from a particular human immunoglobulin that is usuallyhomologous to the non-human donor immunoglobulin to be humanized, or touse a consensus framework from many human antibodies. The secondcriterion is to use the donor amino acid rather than the acceptor if thehuman acceptor residue is unusual and the donor residue is typical forhuman sequences at a specific residue of the framework. The thirdcriterion is to use the donor framework amino acid residue rather thanthe acceptor at positions immediately adjacent to the CDRs. The fourthcriterion is to use the donor amino acid residue at framework positionsat which the amino acid is predicted to have a side chain atom withinabout 3 Å of the CDRs in a three-dimensional immunoglobulin model and tobe capable of interacting with the antigen or with the CDRs of thehumanized immunoglobulin. It is proposed that criteria two, three orfour may be applied in addition or alternatively to criterion one, oreach criteria may be applied singly or in any combination.

In addition, WO 90/07861 [18] details the preparation of a singleCDR-grafted humanized antibody, a humanized antibody specificity for thep55 Tac protein of the IL-2 receptor, by employing the combination ofall four criteria, as above, in designing this humanized antibody. Thevariable region frameworks of the human antibody EU (see, Kabat et al.,1991 [15]) were used as acceptor. In the resultant humanized antibody,the donor CDRs were as defined by Kabat et al., 1991 [15] and Wu et al.,1970 [16] and, in addition, the mouse donor residues were used in placeof the human acceptor residues, at positions 27, 30, 48, 66, 67, 89, 91,94, 103, 104, 105 and 107 in heavy chain and at positions 48, 60 and 63in the light chain, of the variable region frameworks. The humanizedanti-Tac antibody obtained was reported to have an affinity for p55 of3×10⁹ M⁻¹, about one-third of that of the murine MAb.

Several other groups have demonstrated that Queen et al.'s approach offirst choosing homologous frameworks followed by reintroduction of mouseresidues may not be necessary to achieve humanized antibodies withsimilar binding affinities to the original mouse antibodies (Riechmannet al., 1988 [4]; Tempest et al., 1991 [5]; Verhoeyen, et al. 1991[21]). Moreover, these groups have used a different approach and havedemonstrated that it is possible to utilize, as standard, the V regionframeworks derived from NEWM and REI heavy and light chains respectivelyfor CDR-grafting without radical introduction of mouse residues.However, the determination of which mouse residues should be introducedto produce antibodies with binding efficiencies similar to the originalmurine MAb can be difficult to predict, being largely empirical and nottaught by available prior art. In the case of the humanized CAMPATH-IHantibody, the substitution of a phenylalanine for a serine residue atposition 27 was the only substitution required to achieve a bindingefficiency similar to that of the original murine antibody (Riechmann,et al., 1988 [4]; WO92/04381 [22]). In the case of a humanized(reshaped) antibody specific for respiratory syncytial virus (RSV) forthe inhibition of RSV infection in vivo, substitution of a block of 3residues adjacent to CDR3 in the CDR-grafted NEWM heavy chain wasrequired to produce biological activity equivalent to the original mouseantibody (Tempest et al., 1991 [5]; WO 92/04381 [22]). The reshapedantibody in which only the mouse CDRs were transferred to the humanframework showed poor binding for RSV. An advantage of using the Tempestet al., 1991 [5] approach to construct NEWM and REI based humanizedantibodies is that the 3-dimensional structures of NEWM and REI variableregions are known from x-ray crystallography and thus specificinteractions between CDRs and V region framework residues can bemodelled.

Regardless of the approach taken, the examples of the initial humanizedantibodies prepared to date have shown that it is not a straightforwardprocess to obtain humanized antibodies with the characteristics, inparticular, the binding affinity, as well as other desirable properties,of the original murine MAb from which the humanized antibody is derived.Regardless of the approach to CDR grafting taken, it is often notsufficient merely to graft the CDRs from a donor Ig onto the frameworkregions of an acceptor Ig (see, e.g., Tempest et al., 1991 [5],Riechmann et al., 1988 [4], etc., cited herein). In a number of cases,it appears to be critical to alter residues in the framework regions ofthe acceptor antibody in order to obtain binding activity. However, evenacknowledging that such framework changes may be necessary, it is notpossible to predict, on the basis of the available prior art, which, ifany, framework residues will need to be altered to obtain functionalhumanized recombinant antibodies of the desired specificity. Resultsthus far indicate that changes necessary to preserve specificity and/oraffinity are for the most part unique to a given antibody and cannot bepredicted based on the humanization of a different antibody.

In particular, the sets of residues in the framework region which areherein disclosed as being of critical importance to the activity of therecombinant humanized anti-VLA-4 antibodies constructed in accordancewith the teachings of the present invention do not generally coincidewith residues previously identified as critical to the activity of otherhumanized antibodies and were not discovered based on the prior art.

C. Therapeutic Applications of Humanized Antibodies

To date, humanized recombinant antibodies have been developed mainly fortherapeutic application in acute disease situations (Tempest, et al.,1991 [5]) or for diagnostic imaging (Verhoeyen, et al., 1991 [21]).Recently, clinical studies have begun with at least two humanizedantibodies with NEWM and REI V region frameworks, CAMPATH-IH (Riechmannet al., 1988 [4]) and humanized anti-placental alkaline phosphatase(PLAP) (Verhoeyen et al., 1991 [21]) and these studies have initiallyindicated the absence of any marked immune reaction to these antibodies.A course of treatment with CAMPATH-IH provided remission for twopatients with non-Hodgkin's lymphoma thus demonstrating efficacy in achronic disease situation (Hale et al., 1988 [23]). In addition, thelack of immunogenicity of CAMPATH-1H was demonstrated after dailytreatment of the two patients for 30 and 43 days. Since good toleranceto humanized antibodies has been initially observed with CAMPATH-IH,treatment with humanized antibody holds promise for the prevention ofacute disease and to treatment of diseases with low mortality.

D. The VCAM-VLA-4 Adhesion Pathway and Antibodies to VLA-4

Vascular endothelial cells constitute the lining of blood vessels andnormally exhibit a low affinity for circulating leukocytes (Harlan, 1985[24]). The release of cytokines at sites of inflammation, and inresponse to immune reactions, causes their activation and results in theincreased expression of a host of surface antigens. (Collins et al.,1986 [25]; Pober et al., 1986 [26]; Bevilacqua et al., 1987 [27];Leeuwenberq et al., 1989 [28]). These include the adhesion proteinsELAM-1, which binds neutrophils (Bevilacqua et al., 1989 [29], ICAM-1which interacts with all leukocytes (Dustin et al., 1986 [30]; Pober etal. 1986, [26]; Boyd et al., 1988 [31]; Dustin and Springer, 1988 [32]),and VCAM-1 which binds lymphocytes (Osborn et al., 1989 [33]). Thesecytokine-induced adhesion molecules appear to play an important role inleukocyte recruitment to extravascular tissues.

The integrins are a group of cell-extracellular matrix and cell-celladhesion receptors exhibiting an alpha-beta heterodimeric structure,with a widespread cell distribution and a high degree of conservationthroughout evolution (Hynes, 1987 [34]; Marcantonio and Hynes, 1988[35]). The integrins have been subdivided into three major subgroups;the β₂ subfamily of integrins (LFA-1, Mac-1, and p150, 95) is mostlyinvolved in cell-cell interactions within the immune system (Kishimotoet al., 1989 [36]), whereas members of the β₁ and β₃ integrinsubfamilies predominantly mediate cell attachment to the extracellularmatrix (Hynes, 1987 [34]; Ruoslahti, 1988 [37]). In particular, the β₁integrin family, also termed VLA proteins, includes at least sixreceptors that specifically interact with fibronectin, collagen, and/orlaminin (Hemler, 1990 [38]). Within the VLA family, VLA-4 is atypicalbecause it is mostly restricted to lymphoid and myeloid cells (Hemler etal., 1987 [39]), and indirect evidence had suggested that it might beinvolved in various cell-cell interactions (Clayberger et al., 1987[40]; Takada et al., 1989 [41]; Holtzmann et al., 1989 [42]; Bendarczykand McIntyre, 1990 [43]). In addition, VLA-4 has been shown to mediate Tand B lymphocyte attachment to the heparin II binding fragment of humanplasma fibronectin (FN) (Wayner et al., 1989 [44]).

VCAM-1, like ICAM-1, is a member of the immunoglobulin gene superfamily(Osborn et al., 1989 [33]). VCAM-1 and VLA-4 were demonstrated to be aligand-receptor pair that allows attachment of lymphocytes to activatedendothelium by Elices et al., 1990 [45]. Thus, VLA-4 represents asingular example of a β₁ integrin receptor participating in bothcell-cell and cell-extracellular matrix adhesion functions by means ofthe defined ligands VCAM-1 and FN.

VCAM1 (also known as INCAM-110) was first identified as an adhesionmolecule induced on endothelial cells by inflammatory cytokines (TNF andIL-1) and LPS (Rice et al., 1989 [46]; Osborn et al., 1989 [33]).Because VCAM1 binds to cells exhibiting the integrin VLA-4 (α₄β₁)including T and B lymphocytes, monocytes, and eosinophils, but notneutrophils, it is thought to participate in recruitment of these cellsfrom the bloodstream to areas of infection and inflammation (Elices etal, 1990 [45]; Osborn, 1990 [33]). The VCAM1/VLA-4 adhesion pathway hasbeen associated with a number of physiological and pathologicalprocesses. Although VLA-4 is normally restricted to hematopoieticlineages, it is found on melanoma cell lines, and thus it has beensuggested that VCAM1 may participate in metastasis of such tumors (Riceet al., 1989 [46]).

In vivo, VCAM1 is found on areas of arterial endothelium representingearly atherosclerotic plaques in a rabbit model system (Cybulsky andGimbrone, 1991 [47]). VCAM1 is also found on follicular dendritic cellsin human lymph nodes (Freedman et al., 1990 [48]). It is also present onbone marrow stromal cells in the mouse (Miyake et al., 1991 [49]), thusVCAM1 appears to play a role in B-cell development.

The major form of VCAM1 in vivo on endothelial cells, has been referredto as VCAM-7D, and has seven Ig homology units or domains; domains 4, 5and 6 are similar in amino acid sequence to domains 1, 2 and 3,respectively, suggesting an intergenic duplication event in theevolutionary history of the gene (Osborn et al., 1989 [33]; Polte et al.1990 [50]; Hession et al., 1991 [51]; Osborn and Benjamin, U.S. Ser. No.07/821,712 filed Sep. 30, 1991, [52]). A 6-domain form (referred to asVCAM-6D herein) is generated by alternative splicing, in which thefourth domain is deleted (Osborn et al., 1989 [33]; Hession et al. 1991[51], Cybulsky et al., 1991 [47]; Osborn and Benjamin, U.S. Ser. No.07/821,712 filed Sep. 30, 1991 [52]). The VCAM-6D, was the firstsequenced of these alternate forms, however, later in vivo studiesshowed that the VCAM-7D form was dominant in vivo. The biologicalsignificance of the alternate splicing is not known, however as shown byOsborn and Benjamin, U.S. Ser. No. 07/821,712 filed Sep. 30, 1991 [52],VCAM-6D can bind VLA-4-expressing cells and thus clearly has potentialfunctionality in vivo.

The apparent involvement of the VCAM1/VLA-4 adhesion pathway ininfection, inflammation and possibly atherosclerosis has led tocontinuing intensive research to understand the mechanisms of cell-celladhesion on a molecular level and has led investigators to proposeintervention in this adhesion pathway as a treatment for diseases,particularly inflammation (Osborn et al., 1989 [33]). One method ofintervention in this pathway could involve the use of anti-VLA-4antibodies.

Monoclonal antibodies that inhibit VCAM 1 binding to VLA-4 are known.For example, anti-VLA-4 MAbs HP2/1 and HP1/3 have been shown to blockattachment of VLA-4-expressing Ramos cells to human umbilical vein cellsand VCAM1-transfected COS cells (Elices et al., 1990 [45]). Also,anti-VCAM1 antibodies such as the monoclonal antibody 4B9 (Carlos etal., 1990 [53]) have been shown to inhibit adhesion of Ramos(B-cell-like), Jurkat (T-cell-like) and HL60 (granulocyte-like) cells toCOS cells transfected to express VCAM-6D and VCAM-7D (Hession et al.,1991 [51]).

The monoclonal antibodies to VLA-4 that have been described to date fallinto several categories based on epitope mapping studies (Pulido, etal., 1991 [54]). Importantly one particular group of antibodies, toepitope “B”, are effective blockers of all VLA-4-dependent adhesivefunctions (Pulido et al., 1991, [54]). The preparation of suchmonoclonal antibodies to epitope B of VLA 4, including, for example theHP1/2 MAb, have been described by Sanchez-Madrid et al., 1986, [55].Antibodies having similar specificity and having high binding affinitiesto VLA-4 comparable to that of HP1/2, would be particularly promisingcandidates for the preparation of humanized recombinant anti-VLA-4antibodies useful as assay reagents, diagnostics and therapeutics.

As stated above, inflammatory leukocytes are recruited to sites ofinflammation by cell adhesion molecules that are expressed on thesurface of endothelial cells and which act as receptors for leukocytesurface proteins or protein complexes. In particular, eosinophils haverecently been found to participate in three distinct cell adhesionpathways to vascular endothelium, binding to cells expressingintercellular adhesion molecule-1 (ICAM-1), endothelial cell adhesionmolecule-1 (ELAM-1), and vascular cell adhesion molecule-1 (VCAM-1)(Weller et al., 1991 [56]; Walsh et al., 1991 [57]; Bochner et al., 1991[58]; and Dobrina et al., 1991 [59]). That eosinophils express VLA-4differentiates them from other inflammatory cells such as neutrophils,which bind to ELAM-1 and ICAM-1 but not VCAM-1.

The VLA-4-mediated adhesion pathway has been investigated in an asthmamodel to examine the possible role of VLA-4 in leukocyte recruitment toinflamed lung tissue (Lobb, U.S. Ser. No. 07/821,768 filed Jan. 13, 1992[60]). Administering anti-VLA-4 antibody inhibited both the late phaseresponse and airway hyperresponsiveness in allergic sheep. Surprisingly,administration of anti-VLA-4 led to a reduction in the number of bothneutrophils and eosinophils in the lung at 4 hours after allergenchallenge, even though both cells have alternate adhesion pathways bywhich they can be recruited to lung tissues. Also surprisingly,inhibition of hyperresponsiveness in the treated sheep was observedwhich continued to 1 week, even though infiltration of leukocytes,including neutrophils and eosinophils, was not significantly reducedover time.

The VLA-4-mediated adhesion model has also been investigated in aprimate model of inflammatory bowel disease (IBD) (Lobb, U.S. Ser. No,07/835,139 filed Feb. 12, 1992 [61]). The administration of anti-VLA-4antibody surprisingly and significantly reduced acute inflammation inthat model, which is comparable to ulcerative colitis in humans.

More recently, anti-VLA-4 antibodies have been used in methods for theperipheralizing of CD34+ cells, including hematopoietic stem cells asdescribed in Papayannopoulou, U.S. Ser. No. 07/977,702, filed Nov. 13,1992 [62].

Thus, anti-VLA-4 antibodies having certain epitopic specificities andcertain binding affinities may be therapeutically useful in a variety ofinflammatory conditions, including asthma and IBD. In particular,humanized recombinant versions of such anti-VLA-4 antibodies, if theycould be constructed, might be especially useful for administration inhumans. Such humanized antibodies would have the desired potency andspecificity, while avoiding or minimizing an immunological responsewhich would render the antibody ineffective and/or give rise toundesirable side effects.

SUMMARY OF THE INVENTION

The present invention provides a method of constructing a recombinantanti-VLA-4 antibody molecule. Specifically, recombinant antibodiesaccording to the present invention comprise the antigen binding regionsderived from the heavy and/or light chain variable regions of ananti-VLA-4 antibody.

The present invention provides a method for the construction ofhumanized recombinant antibody molecule using as a first step CDRgrafting or “reshaping” technology. Specifically, the humanizedantibodies according to the present invention have specificity for VLA-4and have an antigen binding site wherein at least one or more of thecomplementarity determining regions (CDRs) of the variable domains arederived from a donor non-human anti-VLA-4 antibody, and in which theremay or may not have been minimal alteration of the acceptor antibodyheavy and/or light variable framework region in order to retain donorantibody binding specificity. Preferably, the antigen binding regions ofthe CDR-grafted heavy chain variable domain comprise the CDRscorresponding to positions 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3).Preferably, the antigen binding regions of the CDR-grafted light chainvariable domain comprise CDRs corresponding to positions 24-34 (CDR1),50-56 (CDR2) and 89-97 (CDR3). These residue designations are numberedaccording to the Kabat numbering (Kabat et al., 1991 [15]). Thus, theresidue/position designations do not always correspond directly with thelinear numbering of the amino acid residues shown in the sequencelisting. In the case of the humanized V_(K) sequence disclosed herein,the Kabat numbering does actually correspond to the linear numbering ofamino acid residues shown in the sequence listing. In contrast, in thecase of the humanized V_(H) sequences disclosed herein, the Kabatnumbering does not correspond to the linear numbering of amino acidresidues shown in the sequence listing (e.g., for the humanized V_(H)regions disclosed in the sequence listing, CDR2=50-66, CDR3=99-110).

The invention further provides the recombinant and humanized anti-VLA-4antibodies which may be detectably labelled.

The invention additionally provides a recombinant DNA molecule capableof expressing the recombinant and humanized anti-VLA-4 antibodies of thepresent invention.

The invention further provides host cells capable of producing therecombinant and humanized anti-VLA-4 antibodies of the presentinvention.

The invention additionally relates to diagnostic and therapeutic usesfor the recombinant and humanized anti-VLA-4 antibodies of the presentinvention.

The invention further provides a method for treating inflammationresulting from a response of the specific defense system in a mammaliansubject, including humans, which comprises providing to a subject inneed of such treatment an amount of an anti-inflammatory agentsufficient to suppress the inflammation wherein the anti-inflammatoryagent is a recombinant and humanized anti-VLA-4 antibody of the presentinvention.

The invention further provides a method for treating non-specificinflammation in a mammalian subject, including humans using therecombinant and humanized anti-VLA-4 antibodies.

The invention further concerns the embodiment of the above-describedmethods wherein the recombinant and humanized anti-VLA-4 antibodies ofthe present invention are derived from the murine monoclonal antibodyHP1/2.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The technology for producing monoclonal antibodies is well known.Briefly, an immortal cell line (typically myeloma cells) is fused tolymphocytes (typically splenocytes) from a mammal immunized with wholecells expressing a given antigen, e.g., VLA-4, and the culturesupernatants of the resulting hybridoma cells are screened forantibodies against the antigen (see, generally, Kohler et al., 1975[1]).

Immunization may be accomplished using standard procedures. The unitdose and immunization regimen depend on the species of mammal immunized,its immune status, the body weight of the mammal, etc. Typically, theimmunized mammals are bled and the serum from each blood sample isassayed for particular antibodies using appropriate screening assays.For example, anti-VLA-4 antibodies may be identified byimmunoprecipitation of ¹²⁵I-labeled cell lysates from VLA-4-expressingcells (see, Sanchez-Madrid et al., 1986 [55] and Hemler et al., 1987[39]). Anti-VLA-4 antibodies may also be identified by flow cytometry,e.g., by measuring fluorescent staining of Ramos cells incubated with anantibody believed to recognize VLA-4 (see, Elices et al., 1990 [45]).The lymphocytes used in the production of hybridoma cells typically areisolated from immunized mammals whose sera have already tested positivefor the presence of anti-VLA-4 antibodies using such screening assays.

Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. Preferred immortalcell lines are mouse myeloma cell lines that are sensitive to culturemedium containing hypoxanthine, aminopterin and thymidine (“HATmedium”).

Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using 1500 molecular weight polyethylene glycol (“PEG1500”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridomas producing a desired antibody are detected byscreening the hybridoma culture supernatants. For example, hybridomasprepared to produce anti-VLA-4 antibodies may be screened by testing thehybridoma culture supernatant for secreted antibodies having the abilityto bind to a recombinant α₄-subunit-expressing cell line, such astransfected K-562 cells (see, e.g., Elices et al., 1990 [45]).

To produce anti VLA-4-antibodies, hybridoma cells that tested positivein such screening assays are cultured in a nutrient medium underconditions and for a time sufficient to allow the hybridoma cells tosecrete the monoclonal antibodies into the culture medium. Tissueculture techniques and culture media suitable for hybridoma cells arewell known. The conditioned hybridoma culture supernatant may becollected and the anti-VLA-4 antibodies optionally further purified bywell-known methods.

Alternatively, the desired antibody may be produced by injecting thehybridoma cells into the peritoneal cavity of an unimmunized mouse. Thehybridoma cells proliferate in the peritoneal cavity, secreting theantibody which accumulates as ascites fluid. The antibody may beharvested by withdrawing the ascites fluid from the peritoneal cavitywith a syringe.

Several anti-VLA-4 monoclonal antibodies have been previously described(see, e.g., Sanchez-Madrid et al., 1986 [55]; Hemler et al., 1987 [39];Pulido et al., 1991 [54]). HP1/2, for example, is one such murinemonoclonal antibody which recognizes VLA-4. VLA-4 acts as a leukocytereceptor for plasma fibronectin and VCAM-1. Other monoclonal antibodies,such as HP2/1, HP2/4, L25 and P4C2, have been described that alsorecognize VLA-4.

Recombinant antibodies have been constructed and are described herein inwhich the CDRs of the variable domains of both heavy and light chainswere derived from the murine HP1/2 sequence. Preferred startingmaterials for constructing recombinant humanized antibodies according tothe present invention are anti-VLA-4 antibodies, such as HP1/2, thatblock the interaction of VLA-4 with both VCAM1 and fibronectin.Particularly preferred are those antibodies, such as HP1/2, which inaddition, do not cause cell aggregation. Some anti-VLA-4 blockingantibodies have been observed to cause such aggregation. The HP1/2 MAb(Sanchez-Madrid et al., 1986 [55]) is a particularly excellent candidatefor humanization since it has an extremely high potency, blocks VLA-4interaction with both VCAM1 and fibronectin, but does not cause cellaggregation, and has the specificity for epitope B on VLA-4. In theinitial experiments, V_(H) and V_(K) DNA were isolated and cloned froman HP1/2-producing hybridoma cell line. The variable domain frameworksand constant domains for humanization were initially derived from humanantibody sequences.

The three CDRs that lie on both heavy and light chains are composed ofthose residues which structural studies have shown to be involved inantigen binding. Theoretically, if the CDRs of the murine HP1/2 antibodywere grafted onto human frameworks to form a CDR-grafted variabledomain, and this variable domain were attached to human constantdomains, the resulting CDR-grafted antibody would essentially be a humanantibody with the specificity of murine HP1/2 to bind human VLA-4. Giventhe highly “human” nature of this antibody, it would be expected to befar less immunogenic than murine HP1/2 when administered to patients.

However, following testing for antigen binding of a CDR-grafted HP1/2antibody in which only the CDRs were grafted onto the human framework,it was shown that this did not produce a CDR-grafted antibody havingreasonable affinity for the VLA-4 antigen. It was therefore decided thatadditional residues adjacent to some of the CDRs and critical frameworkresidues needed to be substituted from the human to the correspondingmurine HP1/2 residues in order to generate an antibody with bindingaffinity in the range of 10% to 100% of the binding affinity of themurine HP1/2 MAb. Empirically, changes of one or more residues in theframework regions of V_(H) and V_(K) were made to prepare antibodies ofthe desired specificity and potency, but without making so many changesin the human framework so as to compromise the essentially human natureof the humanized V_(H) and V_(K) region sequences.

Furthermore, VLA-4-binding fragments may be prepared from therecombinant anti-VLA-4 antibodies described herein, such as Fab, Fab′,F(ab′)₂, and F(v) fragments; heavy chain monomers or dimers; light chainmonomers or dimers; and dimers consisting of one heavy chain and onelight chain are also contemplated herein. Such antibody fragments may beproduced by chemical methods, e.g., by cleaving an intact antibody witha protease, such as pepsin or papain, or via recombinant DNA techniques,e.g., by using host cells transformed with truncated heavy and/or lightchain genes. Heavy and light chain monomers may similarly be produced bytreating an intact antibody with a reducing agent such as dithiothreitolor β-mercaptoethanol or by using host cells transformed with DNAencoding either the desired heavy chain or light chain or both.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to belimiting in nature. In the following examples, the necessary restrictionenzymes, plasmids, and other reagents and materials may be obtained fromcommercial sources and cloning, ligation and other recombinant DNAmethodology may be performed by procedures well-known in the art.

Example 1 Isolation of DNA Sequences Encoding Murine Anti-VLA-4 VariableRegions

A. Isolation of the HP1/2 Heavy and Light Chain cDNA

To design a humanized recombinant antibody with specificity for VLA-4,it was first necessary to determine the sequence of the variable domainof the murine HP1/2 heavy and light chains. The sequence was determinedfrom heavy and light chain cDNA that had been synthesized fromcytoplasmic RNA according to methods referenced in Tempest et al., 1991[5].

1. Cells and RNA Isolation

Cytoplasmic RNA (˜200 μg) was prepared by the method of Favaloro et al.,1980 [63], from a semi-confluent 150 cm2 flask of HP1/2-producinghybridoma cells (about 5×105 logarithmic phase cells). The cells werepelleted and the supernatant was assayed for the presence of antibody bya solid phase ELISA using an Inno-Lia mouse monoclonal antibodyisotyping kit (Innogenetics, Antwerp, Belgium) using both the kappaconjugate and the lambda conjugate. The antibody was confirmed to beIgG1/_(K) by this method.

2. cDNA Synthesis

cDNAs were synthesized from the HP1/2 RNA via reverse transcriptioninitiated from primers based on the 5′ end of either the murine IgG1 CH₁or the murine kappa constant domains using approximately 5 μg RNA and 25pmol primer in reverse transcriptase buffer containing 1μ 1/50 μlPharmacia (Milton Keynes, United Kingdom) RNA Guard™ and 250 micromolardNTPs. The sequence of these primers, CG1FOR and CK2FOR are shown as SEQID NO: 1 and SEQ ID NO: 2, respectively. The mixture was heated to 70°C., then allowed to cool slowly to room temperature. Then, 100 units/50μl MMLV reverse transcriptase (Life Technologies, Paisley, UnitedKingdom) was added and the reaction was allowed to proceed at 42° C. forone hour.

3. Amplification of V_(H) and V_(K) cDNA

Polymerase chain reaction (PCR) of murine MAb variable regions can beachieved using a variety of procedures, for example, anchored PCR orprimers based on conserved sequences (see, e.g., Orlandi et al., 1989[64]). Orlandi et al. [64], Huse et al., 1989 [65] and Jones and Bendig,1991 [66], have described some variable region primers. We have beenunsuccessful, however, in using a number of such primers, particularlythose for the light chain PCR of HP1/2 derived V_(K) sequences.

HP1/2 Ig V_(H) and V_(K) cDNAs were amplified by PCR as described bySaiki et al., 1988 [67] and Orlandi et al., 1989 [64]. Reactions werecarried out using 2.5 units/50 ul Amplitaq™ polymerase (Perkin ElmerCetus, Norwalk, Conn.) in 25 cycles of 94° C. for 30 seconds followed by55° C. for 30 seconds and 75° C. for 45 seconds. The final cycle wasfollowed by five minute incubation at 75° C. The same 3′oligonucleotides used for cDNA synthesis were used in conjunction withappropriate 5′ oligonucleotides based on consensus sequences ofrelatively conserved regions at the 5′ end of each V region. V_(H) cDNAwas successfully amplified using the primers VH1BACK [SEQ ID NO: 3] andCG1FOR [SEQ ID NO: 1] and yielded an amplification product ofapproximately 400 bp. V_(K) cDNA was successfully amplified using theprimers VK5BACK [SEQ ID NO: 4] and CK2FOR [SEQ ID NO: 2] and yielded anamplification product of approximately 380 bp.

4. Cloning and Sequencing V_(H) DNA

The primers used for the amplification of V_(H) DNA, contain therestriction enzyme sites PstI and HindIII which facilitate cloning intosequencing vectors. The general cloning and ligation methodology was asdescribed in Molecular Cloning A Laboratory Manual 1982, [68]. Theamplified DNA was digested with PstI to check for internal PstI sitesand an internal PstI site was found. Therefore, the VH DNA was cloned asPstI-PstI and PstI-HindIII fragments into M13 mp18 and 19. The resultingcollection of clones from two independent cDNA preparations weresequenced by the dideoxy method (Sanger, et al., 1977, [69] usingSequenase™ (United States Biochemicals, Cleveland, Ohio, USA). Thesequence of a region of −100-250 bp was determined from each of 25clones. Out of more than 4000 nucleotides sequenced, there were threePCR-induced transition mutation in three separate clones. The HP1/2V_(H) DNA sequence and its translated amino acid sequence are set forthin SEQ ID NO: 5 and SEQ ID NO: 6, respectively. It should be noted thatthe first eight amino acids are dictated by the 5′ primer used in thePCR. Computer-assisted comparisons indicate that HP1/2 VH [SEQ ID NOS: 5and 6] is a member of family IIC (Kabat et al., 1991, [15]. A comparisonbetween HP1/2 V_(H) [SEQ ID NOS: 5 and 6] and a consensus sequence offamily IIC revealed that the only unusual residues are at amino acidpositions 80, 98 and 121 (79, 94 and 121 in Kabat numbering). AlthoughTyr 80 is invariant in subgroup IIC other sequenced murine VH regionshave other aromatic amino acids at this position although none have Trp.The majority of human and murine VHs have an arginine residue at Kabatposition 94. The presence of Asp 94 in HP1/2 V_(H) is extremely rare;there is only one reported example of a negatively charged residue atthis position. Proline at Kabat position 113 is also unusual but isunlikely to be important in the conformation of the CDRs because of itsdistance from them. The amino acids making up CDR1 have been found inthree other sequenced murine V_(H) regions. However, CDR2 and CDR3 areunique to HP1/2 and are not found in any other reported murine V_(H).

5. Cloning and Sequencing V_(K) DNA

The primers used for the amplification of VK DNA contain restrictionsites for the enzymes EcoRI and HindIII. The PCR products obtained usingprimers VK1BACK [SEQ ID NO: 7], VK5BACK [SEQ ID NO: 4] and VK7BACK [SEQID NO: 8] were purified and cloned into M13. Authentic kappa sequenceswere obtained only with VK5BACK [SEQ ID NO: 4]. The sequence of a regionof ˜200-350 bp was determined by the dideoxy method (Sanger et al.,1977, [69] using Sequenase™ (United States Biochemicals, Cleveland,Ohio, USA) from each of ten clones from two independent cDNApreparations. Out of more than 2 kb sequenced, there were only twoclones which each contained one PCR-induced transition mutation.

The HP1/2 V_(K) DNA sequence and its translated amino acid sequence areset forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The firstfour amino acids are dictated by the 5′ PCR primer but the rest of thesequence is in total agreement with partial protein sequence data. HP1/2V_(K) is a member of Kabat family V (Kabat et al., 1991 [15]) and has nounusual residues. The amino acids of CDR1 and CDR3 are unique. The aminoacids making up CDR2 have been reported in one other murine V_(K).

Example 2 Design of a CDR-Grafted Anti-VLA-4 Antibody

To design a CDR-grafted anti-VLA-4 antibody, it was necessary todetermine which residues of murine HP1/2 comprise the CDRs of the lightand heavy chains.

Three regions of hypervariability amid the less variable frameworksequences are found on both light and heavy chains (Wu and Kabat, 1970[16]; Kabat et al., 1991 [15]). In most cases these hypervariableregions correspond to, but may extend beyond, the CDR. The amino acidsequences of the murine HP1/2 V_(H) and V_(K) chains are set forth inSEQ ID NO: 6 and SEQ ID NO: 10, respectively. CDRs of murine HP1/2 wereelucidated in accordance with Kabat et al., 1991 [15] by alignment withother V_(H) and V_(K) sequences. The CDRs of murine HP1/2 V_(H) wereidentified and correspond to the residues identified in the humanizedV_(H) sequences disclosed herein as follows:

CDR1 AA₃₁-AA₃₅ CDR2 AA₅₀-AA₆₆ CDR3 AA₉₉-AA₁₁₀These correspond to AA₃₁-AA₃₅, AA₅₀-AA₆₅, and AA₉₅-AA₁₀₂, respectively,in Kabat numbering. The CDRs of murine HP1/2 V_(K) were identified andcorrespond to the residues identified in the humanized V_(K) sequencesdisclosed herein as follows:

CDR1 AA₂₄-AA₃₄ CDR2 AA₅₀-AA₅₆ CDR3 AA₈₉-AA₉₇These correspond to the same numbered amino acids in Kabat numbering.Thus, only the boundaries of the V_(K), but not V_(H), CDRs correspondedto the Kabat CDR residues. The human frameworks chosen to accept theHP1/2 CDRs were NEWM and REI for the heavy and light chainsrespectively. The NEWM and the REI sequences have been published inKabat et al., 1991 [15].

An initial stage of the humanization process may comprise the basic CDRgrafting with a minimal framework change that might be predicted fromthe literature. For example, in Riechmann et al., 1988 [4], the MAbCAMPATH-1H was successfully humanized using direct CDR grafting withonly one framework change necessary to obtain an antibody with a bindingefficiency similar to that of the original murine antibody. Thisframework change was the substitution of a Phe for a Ser at position 27.However, using the same humanization strategy by CDR grafting and thesingle framework change discovered by Riechmann et al., 1988 [4] for thepreparation of humanized antibodies having other specificities did notyield antibodies with affinities comparable to the murine antibodiesfrom which they were derived. In such cases, the humanization processmust necessarily include additional empirical changes to achieve thedesired specificity and potency. Such changes may be related to theunique structure and sequence of the starting murine antibody but arenot predictable based upon other antibodies of different specificity andsequence. For example, analysis of the murine V_(H) amino acid sequencefrom HP1/2 as set forth in SEQ ID NO: 6 as compared with the other knownsequences indicated that residues 79, 94 and 113 (Kabat numbering) wereunusual. Of these, only Asp 94 is likely to be important in CDRconformation. Most V_(H) regions that have been sequenced have anarginine at this position which is able to form a salt bridge with arelatively conserved Asp 101 in CDR3. Because NEWM has an Arg 94 andV_(H) CDR3 of HP1/2 has an Asp 101, there remains the possibility that asalt bridge would form which would not normally occur. The presence of anegatively charged residue at position 94 is very unusual and thereforeit was decided to include the Asp 94 into the putative humanized V_(H).

A chimeric (murine V/human IgG1/_(K)) HP1/2 antibody may be useful, butnot a necessary, intermediate in the initial stages of preparing a CDRgrafted construct because (i) its antigen-binding ability may indicatethat the correct V regions have been cloned; and (ii) it may act as auseful control in assays of the various humanized antibodies prepared inaccordance with the present invention.

For V_(H), an M13 clone containing full-length HP1/2 V_(H) was amplifiedusing VH1BACK [SEQ ID NO: 3] and VH1FOR [SEQ ID NO: 11] which containPstI and BstEII sites respectively at the 5′ and 3′ ends of the V_(H)domain. The amplified DNA was cut with BstEII and partially cut withPstI, full-length DNA purified and cloned into M13VHPCR1 (Orlandi etal., 1989 [64]) which had been cut with PstI and BstEII. For V_(K) anM13 clone containing full-length HP1/2 V_(K) was amplified using VK3BACK[SEQ ID NO: 12] and VK1FOR [SEQ ID NO: 13] to introduce PvuII and BglIIsites respectively at the 5′ and 3′ ends of the V_(K) domain. Theamplified DNA was cut with PvuII and BglII and cloned into M13VKPCR1(Orlandi et al., 1989 [64]) which had been cut with PvuII and BclI.

In sum, the 5′ primers used for the amplification of the murine V_(H)and V_(K) regions contain convenient restriction sites for cloning intoour expression vectors. The 3′ primers used in the PCRs were from theconstant regions. Restriction sites at the 3′ end of the variableregions were introduced into cloned murine variable region genes withPCR primers which introduced BstII or BglII sites in the heavy and light(kappa) variable regions, respectively. Additionally, the V_(H) primerchanged Pro 113 to Ser.

The murine V_(H) and V_(K) DNAs were cloned into vectors containing thegpt and hygromycin resistance genes respectively, such as pSVgpt andpSVhyg as described by Orlandi, et al. [64], and appropriate human IgG1,IgG4 or _(K) constant regions were added, for example, as described byTakahashi et al., 1982 [70], Flanagan and Rabbitts, 1982 [71], andHieter et al., 1980 [72], respectively. The vectors were cotransfectedinto the rat myeloma YB2/0 and mycophenolic acid resistant clonesscreened by ELISA for secretion of chimeric IgG/_(K) antibody. The YB2/0cell line was described by Kilmartin et al., 1982 [73] and is availablefrom the American Type Culture Collection (ATCC, Rockville, Md.). ELISApositive clones were expanded and antibody purified from culture mediumby protein A affinity chromatography. The chimeric antibody purifiedfrom the transfected cells was assayed for anti-VLA-4 antibody activityas described in Example 7 and was found to be equipotent with the murineHP1/2 antibody.

Example 3 Transplantation of CDR Sequences and Mutagenesis of SelectedFramework Residues

Transplantation of the CDRs into human frameworks was performed usingM13 mutagenesis vectors. The human frameworks chosen to accept the CDRsequences outlined in Example 2 were derived from NEWM for V_(H) and REIfor V_(K), each in an M13 mutagenesis vector. The M13 mutagenesisvectors used for V_(H) and V_(K), were M13VHPCR1 and M13VKPCR2,respectively. M13VKPCR2 is identical to M13VKPCR1 as described byOrlandi et al., 1989 [64], except for a single amino acid change fromvaline (GTG) to glutamine (GAA) in framework 4 of the REI V_(K) codingsequence. M13VHPCR1 described by Orlandi et al., 1989 [64] is M13 thatcontains the coding sequence for a V_(H) region that is an NEWMframework sequence with CDRs derived from an anti-hapten(4-hydroxy-3-nitrophenyl acetyl caproic acid) antibody; the irrelevantV_(H) CDRs are replaced by site-directed mutagenesis with the CDRsderived from HP1/2 V_(H) as described below. The V_(H) region sequence(DNA and amino acid) encoded by M13VHPCR1 is shown as SEQ ID NOS: 14 and15. M13VKPCR2, like M13VKPCR1 described by Orlandi et al. [64], is M13that contains the coding sequence for a V_(K) region that is N-terminalmodified REI framework sequence with CDRs derived from an anti-lysozymeantibody; these irrelevant V_(K) CDRs are replaced by site-directedmutagenesis with the CDRs derived from HP1/2 V_(K) as described below.The V_(K) region sequence (DNA and amino acid) encoded by M13PCR2 isshown as SEQ ID NOS: 16 and 17.

Synthetic oligonucleotides were synthesized containing the HP1/2-derivedV_(H) and VK CDRs flanked by short sequences drawn from NEWM and REIframeworks, respectively, and grafted into the human frameworks byoligonucleotide site-directed mutagenesis as follows. For CDR graftinginto the human V_(H) framework, mutagenizing oligonucleotides 598 [SEQID NO: 18], 599 [SEQ ID NO: 19] and 600 [SEQ ID NO: 20] were used. ForCDR grafting into the human V_(K) framework, the mutagenizingoligonucleotides were 605 [SEQ ID NO: 21], 606 [SEQ ID NO: 22] and 607[SEQ ID NO: 23]. To 5 μg of V_(H) or V_(K) single-stranded DNA in M13was added a 2-fold molar excess of each of the three V_(H) or V_(K)phosphorylated oligonucleotides together with flanking primers based onM13 sequences, oligo 10 [SEQ ID NO: 24] for V_(H) and oligo 385 [SEQ IDNO: 25] for V_(K). Primers were annealed to the template by heating to70° C. and slowly cooling to 37° C. The annealed DNA was extended andligated with 2.5 U T7 DNA polymerase (United States Biochemicals) and 1U T4 DNA ligase (Life Technologies) in 10 mM Tris HCl pH 8.0, 5 mMMgCl₂, 10 mM DTT, 1 mM ATP, 250 μM dNTPs in a reaction volume of 50 μlat 16° C. for 1-2 hours.

The newly extended mutagenic strand was preferentially amplified using 1U Vent DNA polymerase (New England Biolabs) and 25 pmol oligo 11 [SEQ IDNO: 26] or oligo 391 [SEQ ID NO: 27] (for V_(H) or V_(K), respectively)in 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris HCl pH 8.8, 2 mM MgSO₄, O.1%Triton X-100, 25 μM dNTPs in a reaction volume of 50 μl and subjectingthe sample to 30 cycles of 94°, 30 s; 50°, 30 s; 75°, 90 s.

A normal PCR was then performed by adding 25 pmol oligo 10 [SEQ ID NO:24] (for V_(H)) or oligo 385 [SEQ ID NO: 25] (for V_(K)) with 10 thermalcycles. The product DNAs were digested with HindIII and BamHI and clonedinto M13 mp19. Single-stranded DNA was prepared from individual plaques,sequenced and triple mutants were identified.

The resulting Stage 1 V_(H) construct with the DNA sequence and itstranslated product set forth in SEQ ID NO: 28 and SEQ ID NO: 29,respectively. In addition to the CDR grafting, the Stage 1 V_(H)construct contained selected framework changes. Just prior to CDR1, ablock of sequences was changed to the murine residues Phe 27, Asn 28,Ile 29 and Lys 30 [compare AA₂₇-AA₃₀ of SEQ ID NO: 29 with that ofmurine V_(H) sequence [SEQ ID NO: 6]]. This included Phe-27 assubstituted in the humanization of the rat CAMPATH1-H antibody(Riechmann et al., 1988 [4]), but then also substitutes the next threeresidues found in the murine sequence. Although these four residues arenot nominally included in CDR1 (i.e., are not hypervariable in the Kabatsense), structurally they are a part of the CDR1 loop (i.e., structuralloop residues), and therefore included empirically as part of CDR1. Inaddition, the change from Arg to Asp at residue 94 was made based on therationale discussed in Example 2. An alignment of the CDR-grafted Stage1 framework sequences as compared with the NEWM framework is shown inTable I. The resulting VK1 (DQL) construct with the DNA sequence and itstranslated product are set forth in SEQ ID NO: 30 and SEQ ID NO: 31,respectively. An alignment of the CDR-grafted VK1 (DQL) frameworksequences as compared with the REI framework is shown in Table II.

The CDR replaced V_(H) (Stage 1) and V_(K) (VK1) genes were cloned inexpression vectors according to Orlandi, et al., 1989 [64] to yield theplasmids termed pHuVHHuIgG1, pHuVHHuIgG4 and pHuVKHuCK. For pHuVHHuIgG1and pHuVHHuIgG4, the Stage 1 V_(H) gene together with the Ig heavy chainpromoter, appropriate splice sites and signal peptide sequences wereexcised from the M13 mutagenesis vector by digestion with HindIII andBamHI, and cloned into an expression vector such as pSVgpt as describedby Orlandi et al. [64], containing the murine Ig heavy chain enhancer,the SV40 promoter, the gpt gene for selection in mammalian cells andgenes for replication and selection in E. coli. A human IgG1 constantregion as described in Takahashi et al., 1982 [70] was then added as aBamHI fragment. Alternatively, a human IgG4 construct region asdescribed by Flanagan and Rabbitts, 1982 [71] is added. The constructionof the pHuVKHuCK plasmid, using an expression vector such as pSVhyg asdescribed by Orlandi et al. [64], was essentially the same as that ofthe heavy chain expression vector except that the gpt gene for selectionwas replaced by the hygromycin resistance gene (hyg) and a human kappachain constant region as described by Hieter, 1980, [72] was added. Thevectors were cotransfected into the rat myeloma YB2/0 and mycophenolicacid resistant clones screened by ELISA for secretion of human IgG/_(K)antibody. The YB2/0 cell line was described by Kilmartin et al., 1982[73] and is available from the American Type Culture Collection (ATCC,Rockville, Md.). ELISA positive clones were expanded and antibodypurified from culture medium by protein A affinity chromatography. Thetransfected cells are assayed for anti-VLA-4 antibody activity asdescribed in Example 7.

Example 4 Modification of a CDR Grafted Antibody

Beyond the stages of design and preparation to yield anti-VLA-4antibodies as described above in Examples 2 and 3, additional stages ofempirical modifications were used to successfully prepare humanizedrecombinant anti-VLA-4 antibodies. The Stage 1 modifications asdescribed in Example 3 were based on our analysis of primary sequenceand experience in attempting to successfully humanize antibodies. Thenext modifications, designated as Stage 2, were empirical, based in parton our analysis of 3D modelling data. For the V_(H) region, furthermodifications, designated Stage 3, were so-called “scanning”modifications empirically made to correct any remaining defects inaffinities or other antibody properties. The modifications that weremade in these several stages were empirical changes of various blocks ofamino acids with the goal of optimizing the affinity and other desiredproperties of humanized anti-VLA-4 antibodies. Not every modificationmade during the various stages resulted in antibodies with desiredproperties.

1. Additional Heavy Chain Modifications

a. Stage 2 Modification

An additional empirical change in the V_(H) framework was made with theuse of computer modelling, to generate a Stage 2 construct with the DNAsequence and its translated product set forth in SEQ ID NO: 32 and SEQID NO: 33, respectively. Using computer modelling of the Stage 1 V_(H)region, we determined to make a single change in the framework for Stage2, namely a substitution of a Ser for Lys at position 75 (Kabatnumbering), that is position 76 in SEQ ID NO: 33. This determination wasin part based on the possibility that Lys-75 might project into CDR1 andalter its conformation. The M13 vector containing the Stage 1 CDRgrafted HuVH, as described in Example 3, was used as template fortwo-step PCR-directed mutagenesis using the overlap/extension method asdescribed by Ho et al., 1989 [74]. In the first step, two separate PCRswere set up, one with an end primer, oligo 10, [SEQ ID NO: 24] and aprimer containing the desired mutation, 684 [SEQ ID NO: 34], and theother with the opposite end primer, oligo 11 [SEQ ID NO: 26], and aprimer, 683 [SEQ ID NO: 35], that is complementary to the firstmutagenic primer. The amplification products of this first pair of PCRswere then mixed together and a second PCR step was carried out usingonly the end primers oligos 10 and 11, SEQ ID NO: 24 and SEQ ID NO: 26,respectively. The mutagenized amplification product of this PCR was thencloned into M13 mp19 and sequenced, and a mutant bearing the Lys to Serchange (Stage 2 or “S mutant”) was identified.

This turned out to be a critical change in the humanized heavy chainderived from HP1/2 (see Example 7). However, this critical change in thepreparation of humanized recombinant anti-VLA-4 antibodies according tothe present invention was not similarly critical in the preparation ofother humanized antibodies. Specifically, using the same rationalizationand analysis as outlined above, a change in that position was not foundto be a beneficial change in the humanization of antibodies of 2different specificities. An alignment of the CDR-grafted Stage 2framework sequences as compared with the NEWM, as well as Stage 1sequences, is shown in Table I.

b. Stage 3 Modifications

Additional empirical changes were made as Stage 3 constructs. In Stage3, a series of 5 different block changes of amino acids, for largelyempirical reasons, were made to try to improve potency. These constructsare designated STAW, KAITAS, SSE, KRS, and AS. All contain the position75 Ser (Kabat numbering) changed in Stage 2 [position 76 of SEQ ID NO:35], with other changes as noted. Each of these constructs was preparedby two-step PCR directed mutagenesis using the overlap/extension methodof Ho et al., 1989 [74], as described for the Stage 2 Ser mutant, above.For STAW, the additional changes were Gln to Thr at position 77, Phe toAla at position 78 and Ser to Trp at position 79 (Kabat numbering).These changes were accomplished using end primers, oligos 10 [SEQ ID NO:24] and 11 [SEQ ID NO: 26] in conjunction with mutagenizing primers 713[SEQ ID NO: 36] and 716 [SEQ ID NO: 37]. The STAW V_(H) DNA sequence andits translated amino acid sequence are set forth in SEQ ID NO: 38 andSEQ ID NO: 39, respectively. KAITAS was prepared with additional changesof Arg to Lys (position 66), Val to Ala (67), Met to He (69), Leu to Thr(70) and Val to Ala (71) (Kabat numbering), using oligos 10 [SEQ ID NO:24] and 11 [SEQ ID NO: 26] in conjunction with oligos 706 [SEQ ID NO:40] and 707 [SEQ ID NO: 41]. The KAITAS V_(H) DNA sequence and itstranslated amino acid sequence are set forth in SEQ ID NO: 42 and SEQ IDNO: 43, respectively. SSE had additional changes of Ala to Ser (84) andAla to Glu (85) (Kabat numbering), effected by oligos 10 and 11 witholigos 768 [SEQ ID NO: 44] and 769 [SEQ ID NO: 45]. The SSE V_(H) DNAsequence and its translated amino acid sequence are set forth in SEQ IDNO: 46 and SEQ ID NO: 47, respectively. KRS had additional changes ofArg to Lys (38) and Pro to Arg (40) (Kabat numbering), from oligos 10[SEQ ID NO: 24] and 11 [SEQ ID NO: 26] with oligos 704 [SEQ ID NO: 48]and 705 [SEQ ID NO: 49]. The KRS V_(H) DNA sequence and its translatedamino acid sequence are set forth in SEQ ID NO: 50 and SEQ ID NO: 51,respectively. AS had additional change Val to Ala at position 24 (Kabatnumbering) from oligos 10 [SEQ ID NO: 24] and 11 [SEQ ID NO: 26] witholigos 745 [SEQ ID NO: 52] and 746 [SEQ ID NO: 53]. The AS V_(H) DNAsequence and its translated amino acid sequence are set forth in SEQ IDNO: 54 and SEQ ID NO: 55, respectively. An alignment of the CDR-graftedStage 3 framework sequences with the NEWM, Stage 0 (see below), Stage 1,and Stage 2 sequences is shown in Table I. Importantly, as shown inExample 7, the potency of STAW and AS humanized antibodies wereimproved, while KAITAS and KRS humanized antibodies were not of betterpotency. This could not be predicted.

c. Reverse (Stage 0) Modifications

The two blocks of changes made to generate Stage 1 at positions 28-30(NIK) and 94 (D) were mutated back to the NEWM sequences at positions28-30 (TFS), 94 (R), or both positions 27-30 (TFS) and 94 (R). Theseconstructs were designated Stage 0-A, 0-B and 0-C, respectively. Each ofthese constructs was prepared by two-step PCR directed mutagenesis usingthe overlap/extension method of Ho et al., 1989 [74], as described forthe Stage 2 Ser mutant, above. Stage 0-A and 0-B were generated fromStage 1; Stage 0-C was generated from Stage 0-A, as follows. For Stage0-A, the change was from Asp to Arg at position 94. This change wasaccomplished using end primers, oligos 10 [SEQ ID NO: 24] and 11 [SEQ IDNO: 26] in conjunction with mutagenizing primers 915 [SEQ ID NO: 56] and917 [SEQ ID NO: 57]. For stage 0-B, the changes were from Asn-Ile-Lys toThr-Phe-Ser at positions 28-30. These changes were accomplished by usingend primers 10 [SEQ ID NO: 24] and 11 [SEQ ID NO: 26] in conjunctionwith mutagenizing primers 918 [SEQ ID NO: 58] and 919 [SEQ ID NO: 59].Finally, for stage 0-C, to the change of Asp to Arg at position 94 inStage 0-A were added the changes were from Asn-Ile-Lys to Thr-Phe-Ser atpositions 28-30. These changes were accomplished with the same endprimers and mutagenizing primers described above for the Stage 0-Bconstruct.

TABLE I HEAVY CHAIN SEQUENCES FR1 NEWM ?VQLXXSGPGLVRPSQTLSLTCTVSGSTFS(SEQ ID NO: 105) Humanized Anti-VLA-4: STAGE O-AQVQLQE........................FNIK (residues 20-49 of SEQ ID N0: 29)STAGE O-B QVQLQE........................F....... (SEQ ID N0: 109)STAGE O-C QVQLQE........................F....... (SEQ ID NO: 109)STAGE 1 QVQLQE........................FNIK(residues 20-49 of SEQ ID N0: 29) STAGE 2QVQLQE........................FNIK (residues 20-49 of SEQ ID N0: 33)STAGE 3 (STAW) QVQLQE........................FNIK(residues 20-49 of SEQ ID N0: 39) (KAITAS)QVQLQE........................FNIK (residues 20-49 of SEQ ID N0: 43)(SSE) QVQLQE........................FNIK(residues 1-30 of SEQ ID N0: 47) (KRS)QVQLQE........................FNIK (residues 20-49 of SEQ ID NO: 51)(AS) QVQLQE........................A.FNIK(residues 20-49 of SEQ ID NO: 55) FR2 NEWMWVRQPPGRGLEWIG (SEQ ID NO: 106) Humanized Anti-VLA-4: STAGE O-A................... (SEQ ID NO: 106) STAGE O-B................... (SEQ ID NO: 106) STAGE O-C................... (SEQ ID NO: 106) STAGE 1 ...................(residues 55-68 of SEQ ID N0: 29) STAGE 2 ...................(residues 55-68 of SEQ ID NO: 33) STAGE 3 (STAW) ...................(residues 55-68 of SEQ ID NO: 39) (KAITAS) ...................(residues 55-68 of SEQ ID NO: 43) (SSE) ...................(residues 36-49 of SEQ ID NO: 47) (KRS) ....K. R...........(residues 55-68 of SEQ ID NO: 51) (AS) ...................(residues 55-68 of SEQ ID NO: 55) FR3 NEWMRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAR (SEQ ID N0: 107) Humanized Anti-VLA-4:STAGE O-A .......................................... (SEQ ID N0: 107)STAGE O-B .........................................D(residues 86-117 OF SEQ ID N0: 29) STAGE O-C.........................................D(residues 86-117 OF SEQ ID NO: 29) STAGE 1.........................................D(residues 86-117 OF SEQ ID NO: 29) STAGE 2........S................................D(residues 86-117 OF SEQ ID NO: 33) STAGE 3 (STAW)........S.TAW...........................D(residues 86-117 OF SEQ ID NO: 39) (KAITAS).KA.ITA.S................................D(residues 86-117 OF SEQ ID NO: 43) (SSE)........S........SE......................D(residues 67-98 OF SEQ ID NO: 47) (KRS)........S................................D(residues 86-117 OF SEQ ID NO: 51) (AS)........S................................D(residues 86-117 OF SEQ ID NO: 55) FR4 NEWM WGQGSLVTVSS (SEQ ID NO: 108)Humanized Anti-VLA-4: STAGE O-A... TT ......... (residues 130-140 OF SEQ ID NO: 29) STAGE O-B... TT ......... (residues 130-140 OF SEQ ID NO: 29) STAGE O-C... TT ......... (residues 130-140 OF SEQ ID NO: 29) STAGE 1... TT ......... (residues 130-140 OF SEQ ID NO: 29) STAGE 2... TT ......... (residues 130-140 OF SEQ ID NO: 33) STAGE 3 (STAW)... TT ......... (residues 130-140 OF SEQ ID NO: 39) (KAITAS)... TT ......... (residues 130-140 OF SEQ ID NO: 43) (SSE)... TT ......... (residues 111-121 OF SEQ ID NO: 47) (KRS)... TT ......... (residues 130-140 OF SEQ ID NO: 51) (AS)... TT ......... (residues 130-140 OF SEQ ID NO: 55) Note: X denotesGlx., ?denotes Q or E.

2. Light Chain Modifications

In our experience, the humanized light chain generally requires few, ifany, modifications. However, in the preparation of humanized anti-VLA-4antibodies, it became apparent that the light chain of HP1/2 did requireseveral empirical changes. For example, humanized heavy chain of theStage 2 construct (the Ser mutant) with murine light chain was about 2.5fold lower potency than murine HP1/2, while the same humanized heavychain with humanized light chain was about 4-fold lower potency. TheStage 1 humanized V_(K) construct was designated VK1 (DQL) and the DNAsequence and its translated amino acid sequence are set forth in SEQ IDNO: 30 and SEQ ID NO: 31, respectively. The DQL mutations arose from thePCR primer used in the initial cloning of the V_(K)region (see Example1). Alterations were made in the light chain, generating two mutants,SVMDY and DQMDY (VK2 and VK3, respectively). The SVMDY mutant wasprepared from the DQL sequence using oligos 10 [SEQ ID NO: 24] and 11[SEQ ID NO: 26] for DY sequences with oligos 697 [SEQ ID NO: 60 and 698[SEQ ID NO: 61] for SVM sequences. The VK2 (SVMDY) DNA sequence and itstranslated amino acid sequence are set forth in SEQ ID NO: 62 and SEQ IDNO: 63, respectively. The DQMDY sequences were restored to the originalREI framework sequences by two-step PCR-directed mutagenesis using endprimers 10 [SEQ ID NO: 24] and 11 [SEQ ID NO: 26] with mutagenic primers803 [SEQ ID NO: 64] and 804 [SEQ ID NO: 65], and using the SVMDYsequence as template. The VK3 (DQMDY) DNA sequence and its translatedamino acid sequence are set forth in SEQ ID NO: 66 and SEQ ID NO: 67,respectively. The change in the amino terminus (SVM versus DQM) is notrelevant, and relates to the amino terminus of the murine light chain.The other two changes, D and Y, were made to improve potency, and didindeed do so as described in Example 7. An alignment of the CDR-graftedDQL (VK1), SVMDY (VK2) and DQMDY (VK3) framework sequences as comparedwith the REI sequence is shown in Table II.

When the AS mutant heavy chain was combined with the improved lightchain (SVMDY), the resulting humanized antibody was equipotent withmurine HP1/2 as shown in Table III.

3. Alternative Humanized V_(H) and V_(K) Regions

Alternatively, a humanized V_(H) region sequence based on HP1/2 V_(H)region [SEQ ID NO: 5] may be prepared. One such alternative isdesignated V_(H)-PDLN. The DNA sequence of PDLN V_(H) and its translatedamino acid sequence are set forth as SEQ ID NO: 68 and SEQ ID NO: 69,respectively.

In addition, an alternative humanized V_(K) region sequence based on theHP1/2 V_(K) region [SEQ ID NO: 9] may be prepared. One such alternativeV_(K) sequence is designated V_(K)-PDLN and its translated amino acidsequence are set forth as SEQ ID NO: 70 and SEQ ID NO: 71, respectively.

The humanized V_(H)-PDLN was prepared by ligating 12 oligonucleotides,which together span the entire humanized variable region, and byscreening for constructs having the correct sequence. The protocol isdescribed in more detail below.

Oligonucleotides 370-119 through 370-130 (SEQ ID NO: 72 through SEQ IDNO: 83, respectively) (20 pmoles each) were dried down, and separatelyresuspended in 20 μl 1× Kinase Buffer containing 1 mM ATP and 1 μl T4polynucleotide kinase (10 U/μl). The kinase reaction mixture wasincubated for 1 hour at 37° C. The reaction was terminated by incubatingat 70° C. for 5 minutes.

The kinase-treated oligonucleotides were combined with each other (240μl total) and ligated together with 26 μl 10 mM ATP and 2 μl T4 DNAligase (10 U/μl), and the reaction mixture was incubated at roomtemperature for 6 hours. The ligation reaction mixture was extractedwith phenol:chloroform (1:1) saturated with TE buffer, and then ethanolprecipitated and washed 5 times with 70% ethanol.

The dried and washed ethanol precipitate was resuspended in 50 μl 1×150mM Restriction Enzyme Buffer (10×150 mM Restriction Enzyme Buffer is 100mM Tris-HCl, pH 8.0, 1.5 M NaCl, 100 mM MgCl₂, 1 mg/ml gelatin, 10 mMdithiothreitol) and incubated with restriction enzymes BstE2 and PstIfor 16 hours at 37° C. The digestion products were electrophoresedthrough a 2% agarose gel, and the band corresponding to 330 bp wasexcised. The fragment was eluted using GENECLEAN II™ and the eluate wasethanol precipitated. The ethanol precipitate was resuspended in 20 μlTE buffer.

Next, the 330 bp fragment was ligated into vector pLCB7 which wasprepared for ligation by digesting with PstI and BstE2,dephosphorylating the 5′ ends with calf alkaline phosphatase,fractionating on a low melting temperature agarose (LMA) gel, andexcising the pLCB7/PstI/BstE2 LMA fragment. The pLCB7 LMA fragment wasthen ligated to the 330 bp oligonucleotide fragment encoding thehumanized V_(H) region using T4 DNA ligase.

The ligation mixture was used to transform E. coli JA221(Iq) toampicillin resistance. Colonies were grown up and mini-prep DNA wasprepared. The recombinant plasmids were screened for the presence of anapproximately 413 bp NotI/BstE2 fragment. DNA sequence analysisidentified vector pMDR1023 as having the designed humanized V_(H)-PDLNsequence.

The humanized V_(K)-PDLN was prepared by ligating 12 oligonucleotides,which together span the entire humanized V_(K)-PDLN variable region, andby screening for constructs having the correct sequence. The protocol isdescribed in more detail below.

Oligonucleotides 370-131 through 370-142 (SEQ ID NO: 84 through SEQ IDNO: 95, respectively) (20 pmoles each) were dried down, and separatelyresuspended in 20 μl 1× Kinase Buffer containing 1 mM ATP and 1 μl T4polynucleotide kinase (10 U/μl). The kinase reaction mixture wasincubated for 1 hour at 37° C. The reaction was terminated by incubatingat 70° C. for 5 minutes.

The kinase-treated oligonucleotides were combined with each other (240μl total) and ligated together with 26 μl 10 mM ATP and 2 μl T4 DNAligase (10 U/μl), and the reaction mixture was incubated at roomtemperature for 6 hours. The ligation reaction mixture was extractedwith phenol:chloroform (1:1) saturated with TE buffer, and then ethanolprecipitated and washed 5 times with 70% ethanol.

The dried and washed ethanol precipitate was resuspended in 40 μl TE,then electrophoresed through a 2% agarose gel, and the bandcorresponding to 380 bp was excised. The fragment was eluted usingGENECLEAN II™ and the eluate was ethanol precipitated. The ethanolprecipitate was resuspended in 20 μl TE buffer.

Next, the 380 bp fragment was ligated into vector pNN03, which wasprepared for ligation by linearizing with HindIII and BamHI,dephosphorylating the 5′ ends with calf alkaline phosphatase,fractionating on a low melting temperature agarose gel, and excising theband corresponding to linearized pNN03 (2.7 kb). The linearized,dephosphorylated pNN03 was then ligated to the 380 bp oligonucleotidefragment encoding the humanized V_(K) region using T4 DNA ligase.

The ligation mixture was used to transform E. coli JA221(Iq) toampicillin resistance. Colonies were grown up and mini-prep DNA wasprepared. The recombinant plasmids were screened for the presence of thevariable region fragment. DNA sequence analysis identified vectorpMDR1025 as having the designed humanized V_(K)-PDLN sequence.

When an antibody with a V_(H)-PDLN containing heavy chain and with aV_(K)-PDLN containing light chain was assayed for potency according toExample 7, the resulting humanized antibody was approximately equipotentwith the murine HP1/2 antibody.

TABLE II LIGHT CHAIN SEQUENCES FR1 REIDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 101) Humanized Anti-VLA-4:Construct VK1 (DQL).....L.................................................(residues 20-42 of  SEQ ID NO: 31) Construct VK2 (SVMDY)S.VM............................................... (residues 20-42 of SEQ ID NO: 63) Construct VK3 (DQMDY)D.QM............................................... (residues 20-42 of SEQ ID NO: 67) FR2 REI WYQQTPGKAPKLLIY (SEQ ID NO: 102)Humanized Anti-VLA-4: VK1 (DQL) .........K...........................(residues 54-68 of  SEQ ID NO: 31) VK2 (SVMDY).........K........................... (residues 54-68 of  SEQ ID NO: 63)VK3 (DQMDY) .........K........................... (residues 54-68 of SEQ ID NO: 67) FR3 REI GVPSRFSGSGSGTDYTFTISSLQPEDIATYYC (SEQ ID NO: 103)Humanized Anti-VLA-4: VK1 (DQL)..........................................F............................(residues 76-107 of  SEQ ID NO: 31) VK2 (SVMDY)........D..........Y...................F............................residues 76-107 of  SEQ ID NO: 63) VK3 (DQMDY)........D..........Y...................F............................(residues 76-107 of  SEQ ID NO: 67) FR4 REI FGQGTKLQIT (SEQ ID NO: 104)Humanized Anti-VLA-4: VK1 (DQL)...............VE.K (residues 117-126 of SEQ ID NO: 31) VK2 (SVMDY)...............VE.K (residues 117-126 of SEQ ID NO: 63) VK3 (DQMDY)...............VE.K (residues 117-126 of SEQ ID NO: 67)

Example 5 Expression of Recombinant Anti-VLA-4 Antibodies

Each of the V_(H) region sequences and V_(K) region sequences preparedaccording to Examples 1-4, are transferred into expression vectors withconstant region sequences, and the vectors are transfected, preferablyvia electroporation, into mammalian cells. The heavy and light chainsequences may be encoded on separate vectors and co-transfected into thecells or alternatively heavy and light chain sequences may be encoded byand transfected as a single vector. Such a single vector will contain 3expression cassettes: one for Ig heavy chain, one for Ig light chain andone for a selection marker. Expression levels of antibody are measuredfollowing transfection, as described below, or as described in Example7.

V_(H) and V_(K) region sequences as described in Example 4, wereinserted into various cloning and expression vectors. For the anti-VLA-4V_(H) region sequences, plasmids containing such sequences [as describedin Examples 1-4] were digested with PstI and BstE2. The plasmid DNAafter digestion with PstI and BstE2, was dephosphorylated andelectrophoresed through 2% agarose gel. The band for ligation wasexcised and the DNA elected using the GENECLEAN™ technique (Bio101,Inc., La Jolla, Calif.), ethanol precipitated and resuspended in 20 μlTE buffer (10 mM Tris-HCl, 1 mM Na₂ EDTA). Then, 10 μl of theresuspended DNA was used for ligation with the PstI/BstE2 digested V_(H)region sequence.

The ligation mixture was used to transform E. coli K 12 JA221 (Iq) toampicillin resistance. E. coli K12 JA221 (Iq) cells have been depositedwith the ATCC (accession number 68845). Recombinant colonies werescreened for the presence of the V_(H) insert. Some of the plasmidscontaining such fragments were sequenced. The V_(H)-containing plasmidswere designated pBAG 184 (V_(H)-STAW), pBAG 183 (V_(H)-KAITAS), pBAG 185(V_(H)-KRS), pBAG 207 (V_(H)-SSE) and pBAG 195 (V_(H)-AS), and weredeposited in E. coli K12 J221 (Iq) cells with the ATCC as accession nos.69110, 69109, 69111, 69116 and 69113, respectively. The plasmidcontaining alternative V_(H)-PDLN region was designated pMDR1023.

For the V_(K) region sequences, the DNA encoding these sequences wereamplified for cloning and transformation using PCR. Prior toamplification, 20 pmoles of each of the V_(K) chain primers were kinasedby incubation with T4 polynucleotide kinase at 37° C. for 60 minutes bya conventional protocol. The kinase reactions were stopped by heating at70° C. for 10 minutes.

The PCR reactions each contained 10 μl 10×PCR buffer (10×PCR buffer is100 mM Tris/HCl, pH 8.3, 500 mM KCl, 15 mM MgCl₂, 0.01% gelatin, 20pmoles each of the appropriate kinased primers, 20 μl cDNA, 0.5 μl Taqpolymerase (5 U/μl, Perkin Elmer-Cetus) and 49.5 μl H₂O. The PCRconditions were 30 cycles of incubation for: 1 minute at 94° C.; 2minutes at 40° C. (for heavy chain PCR) or at 55° C. (for light chainPCR); and 2 minutes at 72° C. For VK1-DQL, primers were 370-247 [SEQ IDNO: 96] and 370-210 [SEQ ID NO: 97]. For VK2-SVMDY, primers were 370-269[SEQ ID NO: 98] and 370-210 [SEQ ID NO: 97]. For VK3-DQMDY, primers were370-268 [SEQ ID NO: 99] and 370-210 [SEQ ID NO: 97].

The reaction mixtures were electrophoresed through 2% agarose gel, andthe bands corresponding to the expected sizes of the light chainvariable region (˜330 bp) were excised with AgeI and BamHI. The DNA inthose bands were eluted using the GENECLEAN™ technique (Bio101 Inc., LaJolla, Calif.), ethanol precipitated and subsequently each resuspendedin 20 μl TE buffer (10 mM Tris-HCl, 1 mM Na₂EDTA).

Klenow fragment of DNA polymerase (New England Biolabs, 5 U/μl) (1 μl)was added to the purified PCR fragments in a reaction volume of 25 μlcontaining 1× ligation buffer (10× ligation buffer is 0.5 M Tris/HCl, pH7.5, 100 mM MgCl2 and 40 mM DTT) and 0.125 mM each of dXTPs and thereaction incubated at room temperature for 15 minutes. The reaction wasterminated by incubation at 70° C. for 5 minutes, and then stored onice.

The fragment from each PCR reaction is ligated to a plasmid such aspNN03 or a plasmid derived from pNN03 such as pLCB7, which had beenpreviously linearized by EcoRV, dephosphorylated and fractionatedthrough low temperature melting agarose. Such plasmids, including pNN03and pLCB7 have been described in co-pending and co-assigned (Burkly etal., U.S. Ser. No. 07/916,098, filed Jul. 24, 1992 [75]).

The ligation mixture was used to transform E. coli K12 JA221(Iq) toampicillin resistance. E. coli K12 JA221(Iq) cells are deposited withAmerican Type Culture Collection (accession number 68845). Recombinantcolonies were screened for the presence of the V_(K) insert. Some of theplasmids containing such fragments were sequenced. The V_(K)-containingplasmids were designated pBAG 190 (VK1-DQL), pBAG 198 (VK2-SVMDY) andpBAG 197 (VK3-DQMDY), and were deposited in E. coli K12 JA 221 (Iq)cells with the ATCC as accession nos. 69112, 69115 and 69114,respectively. The plasmid containing the alternative VK (PDLN) regionwas designated pMDR 1025.

In a series of experiments, the expression vectors encoding recombinantanti-VLA-4 heavy and light chains are transfected via electroporationand the cells are then cultured for 48 hours. After 48 hours of culture,the cells are radiolabelled using ³⁵S-cysteine overnight and then thecell extracts and conditioned media are immunoprecipitated by incubationwith protein A-Sepharose. The protein A-Sepharose is washed and thebound proteins are eluted with SDS-PAGE loading buffer. The samples areanalyzed via electrophoresis through 10% SDS-PAGE gels under reducingconditions. In this way, light chain expression is detected only as aconsequence of the light chains being associated with the heavy chains.The expected sizes of the heavy and light chains as visualized in the10% gels are 50 kD and 25 kD, respectively.

Since recombinant anti-VLA-4 antibody molecules, prepared as describedin Examples 1-4, may be stably expressed in a variety of mammalian celllines, it is possible to express recombinant antibody genes innonsecreting myeloma or hybridoma cell lines under the control ofIg-gene promoters and enhancers or in non-lymphoid cells, such asChinese hamster ovary (CHO) cells, in conjunction with vectoramplification using DHFR selection. Recently, Bebbington et al., 1992[76] have described a method for the high-level expression of arecombinant antibody from myeloma cells using a glutamine synthetasegene as an amplifiable marker. This GS expression system is mostpreferred for the production of recombinant anti-VLA-4 antibodymolecules according to the present invention. The methods, vectors withhCMV promoters and with 5′ untranslated sequences from the hCMV-MIEgenes including cell lines (most preferably NSO) and media for GSexpression of recombinant antibodies is described in detail inBebbington et al., 1992 [76], WO86/05807 [77], WO87/04462 [78],WO89/01036 [79] and WO89/10404 [80].

In accordance with the teachings of these publications, NSO cells weretransfected with a heavy chain sequence having the VH-AS region sequence[SEQ ID NO: 54] and a light chain sequence having the VK-SVMDY sequence[SEQ ID NO: 66] to obtain a stable cell line secreting a humanizedrecombinant anti-VLA-4 antibody with high potency comparable to themurine HP1/2 antibody. This cell line has been deposited with the ATCCon Nov. 3, 1992 and given accession no. CRL 11175. The AS/SVMDYhumanized antibody is at least equipotent with or perhaps more potentthan the murine HP1/2 antibody.

Example 6 Purification of MAbs from Conditioned Media for Assay

To obtain accurate values for half-maximal binding or inhibition, stocksolutions of purified antibodies are needed at known concentrations.Stable cell lines secreting the antibodies of interest were made and thehumanized recombinant anti-VLA-4 antibodies were purified fromconditioned medium using conventional protein A chromatography. Theconcentration of the purified antibodies is assessed by their absorptioncoefficient at 280 nm, which is well established for antibodies.

A cell line producing a humanized anti-VLA-4 antibody is grown in rollerbottles in Dulbecco's modified Eagle medium containing 10% fetal bovineserum. A 2 liter batch of conditioned medium is used for eachpurification run. Cells are removed from the medium by centrifugation ina RC-3B preparative centrifuge (4K, 30 minutes, H4000 rotor) and thesupernatant is filtered first through a 0.45μ membrane and then througha 0.22μ membrane. The medium is stored at 4° C. until it can beprocessed.

Two liters of conditioned medium is concentrated to 220 ml in a spiralultrafiltration unit (Amicon, Corp., Cherry Hill Drive, Danvers, Mass.01923) that is equipped with an S1Y30 (YM30) Diaflo cartridge. Theconcentrate is diluted with 400 ml of protein A binding buffer (3M NaCl,1.5M glycine pH 8.9) and again concentrated to 200 ml. The concentrateis treated in batch with 0.5 ml Fast Flow Protein A Sepharose 4(Pharmacia, Inc., 800 Centennial Avenue, Piscataway, N.J. 08854) using araised stir bar to agitate the mixture. After an overnight incubation at4° C., the resin is collected by centrifugation (5 minutes, 50 g),washed twice with 20 volumes of protein A binding buffer (usingcentrifugation to recover the resin), and transferred to a column forsubsequent treatment. The column is washed four times with 0.5 ml ofprotein A binding buffer, two times with 0.25 ml of PBS, and the IgG iseluted with Pierce IgG elution buffer (Pierce Chemical Co., Rockford,Ill. 61105 Cat No. 21004Y or 21009Y). 180 μl fractions are collected,which are neutralized with 20 μl of 1M HEPES pH 7.5. Fractions areanalyzed for absorptance at 280 nm and by SDS-PAGE. The gel is stainedwith Coomassie blue. Peak fractions are pooled. 100 μl (14 ml/ml) isdiluted with 100 μl of PBS and subjected to gel filtration on a Superose6 FPLC column (Pharmacia, Inc., 800 Centennial Avenue, Piscataway, N.J.08854) in PBS. The column is run at 20 ml/hour and 1.5 minute fractionsare collected. Peak column fractions are pooled, aliquoted, frozen ondry ice, and stored at −70° C. SDS-polyacrylamide gel profile of thefinal product is obtained under reducing and non-reducing conditions. Insome cases when the sample is analyzed under non-reducing conditions,about 10% of the product is not an intact antibody. Studies in thesecases indicate that this product is a heavy-light chain dimer. This hasbeen previously recognized as a problem with IgG4 antibodies.

Example 7 Determination of Relative Binding Affinities of HumanizedRecombinant Anti-VLA-4 Antibodies

Recombinant antibodies according to the present invention are purified,as described in Example 6, and are assayed to determine theirspecificity for VLA-4 and their binding affinity or potency. Inparticular, the potency of a recombinant anti-VLA-4 antibody wasassessed by calculating the half-maximal binding constant (reported asng/ml or μg/ml of purified antibody) using two different assaysdescribed as follows.

1. Inhibition of VLA-4-dependent adhesion to VCAM1

The critical function of an anti-VLA-4 antibody is defined by theability to inhibit the VCAM1/VLA-4 adhesion pathway. It has beenpreviously shown (Lobb et al., 1991a, [81]) that purified recombinantsoluble VCAM1 (rsVCAM1) can be immobilized on plastic and is afunctional adhesion molecule. Immobilized rsVCAM1 binds VLA-4-expressingcells such as the human B cell line Ramos, and this binding can beinhibited by MAbs to VCAM1, such as 4B9 or MAbs to VLA-4, such as HP1/2.This assay provides a reproducible method to assess the potency of anyhumanized recombinant antibody. Briefly, the antibody solution isdiluted, and the serial antibody dilutions are incubated with Ramoscells, which are then incubated with rsVCAM1-coated plates. The Ramoscells are fluorescently labelled as described by Lobb, 1991b [82], andbinding assessed by fluorescence in 96 well cluster plates according tothe following protocol.

Recombinant soluble VCAM1 was prepared and purified essentially asdescribed by Lobb et al., 1991a [81]. Soluble VCAM is diluted to 10μg/ml in 0.05 M NaHCO₃, (15 mM NaHCO₃, 35 mM Na₂CO₃) pH 9.2. Then 50μl/well is added into a Linbro Titertek polystyrene 96 well plate, flatbottom, Flow Labs catalog #76-231-05. The plate is incubated at 4° C.overnight.

Following this incubation, the contents of the wells are removed byinverting and blotting the plate. To the empty wells, 100 μl/well of 1%of BSA in PBS, 0.02% NaN₃ is added for 1 hour or longer at roomtemperature. If the plate is not to be used immediately, it can beblocked and stored for one week at 4° C. BSA is added to some wells toassess non-specific binding.

For binding quantitation, VLA-4 presenting cells, preferably Ramoscells, should be prelabelled. The cells may be radiolabelled orfluorescently labelled. For radiolabelling, prelabelling of the cellsmay be done overnight using ³H-thymidine (0.5 uCi/ml). Alternatively,and preferably, the cells are preincubated with BCECF-AM (chemical name:2′,7′-bis-(2-carboxyethyl)-5(and -6) carboxyfluorescein, acetoxymethylester, Molecular Probes Inc., Eugene, Oreg., catalog #B-1150). For thismethod, cells are suspended to 5×10⁶/ml, 2 μM BCECF-AM is added and themixture is incubated for 30 minutes at 37° C. Following either method,the cells are washed with RPMI, 2% FBS, pH 7.4. RPMI with 1% FBS mayalso be used.

For the binding study, 2-4×10⁶ cells/ml in RPMI, 2% FBS are resuspended,then 50 μl of labelled cells are added per well for 10 minutes ofbinding at room temperature.

After the 10 minute incubation, the contents of the wells are removed byinversion and the plates washed 1-2 times gently with RPMI, 2% FBS. Whenexamined under a light microscope, BSA blank wells should have very fewcells bound. A brief inverted spin may be included to remove cells notfirmly attached and the plates may be washed again 1-2 times.

For the BCECF-AM method, 100 μl of 1% NP40 is added to each well tosolubilize the cells and then the plate is read on a fluorescence platescanner. (If the radiolabelling method is used, 100 μl of 0.1% NaOH isadded to each well and then the contents of each well are transferred toscintillation vials containing cocktail).

A volume of 50 μl of labelled cells should be counted to obtain a totalknown value added to each well. Then the 50 μl of labelled cells areadded to either a well containing only 100 μl of 1% NP40 or to ascintillation vial depending on the method used.

For antibody blocking studies, 100 μl/well of murine HP1/2 MAb(anti-VLA-4) typically at 10 μg/ml in RPMI, 2% FBS are added to thersVCAM1 coated plates and incubated for 30 minutes at room temperatureprior to cell binding as described above. MAb HP1/2 (anti-VLA-4) or anyrecombinant humanized anti-VLA-4 antibody prepared as described hereinmust be preincubated with labelled cells for 30 minutes at roomtemperature prior to the cell binding. Concentrations of the antibodiespreincubated will vary, but generally concentrations were in the rangeof about 1 μg/ml.

In these adhesion assays, murine HP1/2 inhibits Ramos cell bindingcompletely at about 40 ng/ml, and half maximally at about 15 ng/ml (10μM). The results of adhesion assays as represented by the calculatedhalf-maximal binding constants using humanized recombinant anti-VLA-4antibodies made according to the present invention are shown in TableIII. The number (n) of experiments performed for each value is indicatedfor the recombinant humanized antibodies. As discussed below, theseresults generally compare well with the results obtained with the FACSbinding assay.

The potency of recombinant Stage 0, Stage 1, Stage 2 and Stage 3antibodies having the VK1 (DQL) light chain that had been purified fromstably transfected YB2/0 cell lines was measured in the adhesion assay,as shown in Table M. The results showed that there was no inhibitiondetected in concentrations up to 1 μg/ml (1000 ng/ml) with the Stage 0-Band 0-C humanized antibodies. The results with the recombinant Stage 3antibodies STAW and AS having the improved VK2 (SVMDY) light chainshowed that the AS/SVMDY antibody was at least equipotent and perhapsmore potent than the murine HP1/2 antibody. Certain Stage 2 and Stage 3constructs showed potencies of about 20% to about 100% of the potency ofthe murine HP1/2 antibody.

2. FACS Assays

The binding of humanized recombinant antibodies to the cell surface canbe assessed directly by fluorescence activated cell sorter (FACS)analysis, using fluorescently labelled antibodies. This is a standardtechnique that also provides half-maximal binding information followingdose response measurements. The FACS methods are described in Lobb etal., 1991b [82].

Briefly, 25 μl cells (4×106/ml in FACS buffer (PBS 2% FBS, 0.1% NaN₃) onice are added to 5 μl of 5 μg/ml FITC or phycoerythrin (PE) conjugatedantibody in FACS buffer, and incubated in V-bottomed microliter wells onice for 30 minutes. To the wells, 125 μl of FACS buffer is added, theplates are centrifuged at 350×g for 5 minutes, and the supernatant isshaken off. To each well is added 125 μl FACS buffer, then the cells aretransferred to 12×75 mm Falcon polystyrene tubes and resuspended to afinal volume of 250 μl in FACS buffer. The mixture is analyzed on aBecton Dickinson FACS tar. The results of the FACS assays as representedby the calculated half-maximal binding constructs using humanizedrecombinant anti-VLA-4 antibodies made according to the presentinvention are shown in Table III and the number (n) of experimentsperformed for each value is indicated for the humanized antibodies.Table III also shows the potency calculated from the combined adhesionand FACS assays. Murine HP1/2 binds half-maximally to Ramos cells at 15ng/ml. The AS/SVMDY humanized antibody binds half-maximally to Ramoscells at 12 ng/ml. Thus, the two assays (i.e., adhesion and FACS assays)show an excellent correlation for the murine antibody and the humanizedAS/SVMDY antibody.

TABLE III SUMMARY OF HALF-MAXIMAL BINDING CONSTANTS FOR HUMANIZEDRECOMBINANT ANTI-VLA-4 ANTIBODIES Antibody Adhesion Assay FACS AssayCombination Murine HP1/2 15 ng/ml 15 ng/ml 15 ng/ml Stage 0 >1000ng/ml   — — (Humanized (n = 3) heavy chain) Stage 1 228 ng/ml  — 228ng/ml  (Humanized (n = 6) (n = 6) heavy chain) Stage 2 56 ng/ml 47 ng/ml60 ng/ml (Ser mutant) (n = 14) (n = 6) (n = 20) Stage 3 (STAW) 30 ng/ml33 ng/ml 32 ng/ml (n = 3) (n = 3) (n = 6) (KAITAS) 85 ng/ml 100 ng/ml 90 ng/ml (n = 2) (n = 1) (n = 3) (SSE) 100 ng/ml  40 ng/ml 80 ng/ml (n =2) (n = 1) (n = 3) (KRS) 50 ng/ml 70 ng/ml 57 ng/ml (n = 2) (n = 1) (n =3) (AS) 28 ng/ml 14 ng/ml 21 ng/ml (n = 2) (n = 2) (n = 4) Constructswith improved light chain STAW/SVMDY 25 ng/ml 35 ng/ml 29 ng/ml (n = 4)(n = 3) (n = 7) AS/SVMDY 12 ng/ml 12 ng/ml 12 ng/ml (n = 2) (n = 2) (n =4)

Deposits

The following plasmids in E. coli K12 J221 (Iq) cells were depositedunder the Budapest Treaty with American Type Culture Collection (ATCC),Rockville, Md. (USA) on Oct. 30, 1992. The deposits are identified asfollows:

Plasmid Accession No. pBAG 184 (V_(H)-STAW) 69110 pBAG 183(V_(H)-KAITAS) 69109 pBAG 185 (V_(H)-KRS) 69111 pBAG 207 (V_(H)-SSE)69116 pBAG 195 (V_(H)-AS) 69113 pBAG 190 (VK1-DQL) 69112 pBAG 198(VK2-SVMDY) 69115 pBAG 197 (VK3-DQMDY) 69114

In addition, an NSO cell line producing humanized recombinant anti-VLA-4antibody was deposited under the Budapest Treaty with American TypeCulture Collection (ATCC), Rockville, Md. (USA) on Nov. 3, 1992. Thedeposit was given ATCC accession no. CRL 11175.

Sequences

The following is a summary of the sequences set forth in the SequenceListing:

-   SEQ ID NO: 1 DNA sequence of CG1FOR primer-   SEQ ID NO: 2 DNA sequence of CK2FOR primer-   SEQ ID NO: 3 DNA sequence of VH1BACK primer-   SEQ ID NO: 4 DNA sequence of VH5BACK primer-   SEQ ID NO: 5 DNA sequence of HP1/2 heavy chain variable region-   SEQ ID NO: 6 Amino acid sequence of HP1/2 heavy chain variable    region-   SEQ ID NO: 7 DNA sequence of VK1BACK primer-   SEQ ID NO: 8 DNA sequence of VK7BACK primer-   SEQ ID NO: 9 DNA sequence of HP1/2 light chain variable region-   SEQ ID NO: 10 Amino acid sequence of HP1/2 light chain variable    region-   SEQ ID NO: 11 DNA sequence of VH1FOR primer-   SEQ ID NO: 12 DNA sequence of VK3BACK primer-   SEQ ID NO: 13 DNA sequence of VK1FOR primer-   SEQ ID NO: 14 DNA sequence of VH insert in M13VHPCR1-   SEQ ID NO: 15 Amino acid sequence of VH insert in M13VHPCR1-   SEQ ID NO: 16 DNA sequence of VK insert in M13VKPCR2-   SEQ ID NO: 17 Amino acid sequence of VK insert in M13VKPCR2-   SEQ ID NO: 18 DNA sequence of OLIGO598-   SEQ ID NO: 19 DNA sequence of OLIGO599-   SEQ ID NO: 20 DNA sequence of OLIGO600-   SEQ ID NO: 21 DNA sequence of OLIGO605-   SEQ ID NO: 22 DNA sequence of OLIGO606-   SEQ ID NO: 23 DNA sequence of OLIGO607-   SEQ ID NO: 24 DNA sequence of OLIGO10-   SEQ ID NO: 25 DNA sequence of OLIGO385-   SEQ ID NO: 26 DNA sequence of OLIGO11-   SEQ ID NO: 27 DNA sequence of OLIGO391-   SEQ ID NO: 28 DNA sequence of Stage 1 heavy chain variable region-   SEQ ID NO: 29 Amino acid sequence of Stage 1 heavy chain variable    region-   SEQ ID NO: 30 DNA sequence of VK1 (DQL) light chain variable region-   SEQ ID NO: 31 Amino acid sequence of VK1 (DQL) light chain variable    region-   SEQ ID NO: 32 DNA sequence of Stage 2 heavy chain variable region-   SEQ ID NO: 33 Amino acid sequence of Stage 2 heavy chain variable    region-   SEQ ID NO: 34 DNA sequence of OLIGO684-   SEQ ID NO: 35 DNA sequence of OLIGO683-   SEQ ID NO: 36 DNA sequence of OLIGO713-   SEQ ID NO: 37 DNA sequence of OLIGO716-   SEQ ID NO: 38 DNA sequence of STAW heavy chain variable region-   SEQ ID NO: 39 Amino acid sequence of STAW heavy chain variable    region-   SEQ ID NO: 40 DNA sequence of OLIGO706-   SEQ ID NO: 41 DNA sequence of OLIGO707-   SEQ ID NO: 42 DNA sequence of KAITAS heavy chain variable region-   SEQ ID NO: 43 Amino acid sequence of KAITAS heavy chain variable    region-   SEQ ID NO: 44 DNA sequence of OLIGO768-   SEQ ID NO: 45 DNA sequence of OLIGO769-   SEQ ID NO: 46 DNA sequence of SSE heavy chain variable region-   SEQ ID NO: 47 Amino acid sequence of SSE heavy chain variable region-   SEQ ID NO: 48 DNA sequence of OLIGO704-   SEQ ID NO: 49 DNA sequence of OLIGO705-   SEQ ID NO: 50 DNA sequence of KRS heavy chain variable region-   SEQ ID NO: 51 Amino acid sequence of KRS heavy chain variable region-   SEQ ID NO: 52 DNA sequence of OLIGO745-   SEQ ID NO: 53 DNA sequence of OLIGO746-   SEQ ID NO: 54 DNA sequence of AS heavy chain variable region-   SEQ ID NO: 55 Amino acid sequence of AS heavy chain variable region-   SEQ ID NO: 56 DNA sequence of OLIGO915-   SEQ ID NO: 57 DNA sequence of OLIGO917-   SEQ ID NO: 58 DNA sequence of OLIGO918-   SEQ ID NO: 59 DNA sequence of OLIOG919-   SEQ ID NO: 60 DNA sequence of OLIGO697-   SEQ ID NO: 61 DNA sequence of OLIGO698-   SEQ ID NO: 62 DNA sequence of VK2 (SVMDY) light chain variable    region-   SEQ ID NO: 63 Amino acid sequence of VK2 (SVMDY) light chain    variable region-   SEQ ID NO: 64 DNA sequence of OLIGO803-   SEQ ID NO: 65 DNA sequence of OLIGO804-   SEQ ID NO: 66 DNA sequence of VK3 (DQMDY) light chain variable    region-   SEQ ID NO: 67 Amino acid sequence of VK3 (DQMDY) light chain    variable region-   SEQ ID NO: 68 DNA sequence of PDLN heavy chain variable region-   SEQ ID NO: 69 Amino acid sequence of PDLN heavy chain variable    region-   SEQ ID NO: 70 DNA sequence of PDLN light chain variable region-   SEQ ID NO: 71 Amino acid sequence of PDLN light chain variable    region-   SEQ ID NO: 72 DNA sequence of Oligo 370-119-   SEQ ID NO: 73 DNA sequence of Oligo 370-120-   SEQ ID NO: 74 DNA sequence of Oligo 370-121-   SEQ ID NO: 75 DNA sequence of Oligo 370-122-   SEQ ID NO: 76 DNA sequence of Oligo 370-123-   SEQ ID NO: 77 DNA sequence of Oligo 370-124-   SEQ ID NO: 78 DNA sequence of Oligo 370-125-   SEQ ID NO: 79 DNA sequence of Oligo 370-126-   SEQ ID NO: 80 DNA sequence of Oligo 370-127-   SEQ ID NO: 81 DNA sequence of Oligo 370-128-   SEQ ID NO: 82 DNA sequence of Oligo 370-129-   SEQ ID NO: 83 DNA sequence of Oligo 370-130-   SEQ ID NO: 84 DNA sequence of Oligo 370-131-   SEQ ID NO: 85 DNA sequence of Oligo 370-132-   SEQ ID NO: 86 DNA sequence of Oligo 370-133-   SEQ ID NO: 87 DNA sequence of Oligo 370-134-   SEQ ID NO: 88 DNA sequence of Oligo 370-135-   SEQ ID NO: 89 DNA sequence of Oligo 370-136-   SEQ ID NO: 90 DNA sequence of Oligo 370-137-   SEQ ID NO: 91 DNA sequence of Oligo 370-138-   SEQ ID NO: 92 DNA sequence of Oligo 370-139-   SEQ ID NO: 93 DNA sequence of Oligo 370-140-   SEQ ID NO: 94 DNA sequence of Oligo 370-141-   SEQ ID NO: 95 DNA sequence of Oligo 370-142-   SEQ ID NO: 96 DNA sequence of VK1-DQL primer 370-247-   SEQ ID NO: 97 DNA sequence of VK1-DQL primer 370-210-   SEQ ID NO: 98 DNA sequence of VK2-SVMDY primer 370-269-   SEQ ID NO: 99 DNA sequence of VK3-DQMDY primer 370-268

While we have herein before described a number of embodiments of thisinvention, it is apparent that our basic embodiments can be altered toprovide other embodiments that utilize the compositions and processes ofthis invention. Therefore, it will be appreciated that the scope of thisinvention includes all alternative embodiments and variations which aredefined in the foregoing specification and by the claims appendedhereto; and the invention is not to be limited by the specificembodiments that have been presented herein by way of example.

LIST OF REFERENCES CITED

-   [1] Kohler, G. and Milstein, 1975, C. Nature 265:295-497,    “Continuous Cultures of Fused Cells Secreting Antibody of Predefined    Specificity”-   [2] Schroff et al., 1985, Cancer Res 45: 879-885, “Human-antimurine    immunoglobulin responses in patients receiving monoclonal antibody    therapy”-   [3] Borrebaeck et al., 1990, in Therapeutic Monoclonal Antibodies,    Borrebaeck and Larrick (eds.), Stockton Press pp. 1-15-   [4] Riechmann et al., 1988, Nature 332: 323-327, “Reshaping human    antibodies for therapy”-   [5] Tempest et al., 1991, Biotechnology 9: 266-271, “Reshaping a    human monoclonal antibody to inhibit human respiratory syncytial    virus infection in vivo”-   [6] EP 120694 (Celltech Limited)-   [7] EP 125023 (Genentech, Inc. and City of Hope)-   [8] WO 86/01533 (Celltech Limited)-   [9] Begent et al., 1990, Br. J. Cancer 62: 487-   [10] U.S. Pat. No. 4,816,567, Cabilly et al., “Recombinant    Immunoglobin Preparations”, issued Mar. 28, 1989.-   [11] U.S. Pat. No. 4,816,397, Boss et al., “Multichain Polypeptides    Or Proteins And Processes For Their Production”, issued Mar. 28,    1989.-   [12] EP 0239400 (Winter)-   [13] Verhoeyen et al., 1988, Science 239: 1534-1536, “Reshaping of    human antibodies using CDR-grafting in Monoclonal Antibodies”-   [14] WO 89/07454 (Medical Research Council)-   [15] Kabat et al., 1991, 5th Ed., 4 vol., Sequences of Proteins of    Immunological Interest U.S. Department of Health Human Services,    NIH, USA-   [16] Wu et al., 1970, “An Analysis of the Sequences of the Variable    Regions of Bence Jones Proteins and Myeloma Light Chains and Their    Implications for Antibody Complementarity”, J. Exp. Med. 132:    211-250-   [17] Queen et al., 1989, Proc. Natl. Acad. Sci. USA 86: 10029-10033,    “A humanized antibody that binds to the interleukin 2 receptor”-   [18] WO 90/07861 (Protein Design Labs Inc.)-   [19] Co. et al., 1991, Proc. Natl. Acad. Sci. USA 88: 2869-2873,    “Humanised antibodies for antiviral therapy”-   [20] Bruggemann, et al., 1989., J. Exp. Med. 170:2153-2157, “The    immunogenicity of chimeric antibodies”-   [21] Verhoeyen et al., 1991, “Reshaping of Human Antibodies Using    CDR-Grafting” in Monoclonal Antibodies, Chapman and Hall, pp. 37-43.-   [22] WO 92/04881 (Scotgen Limited)-   [23] Hale et al., 1988, “Remission induction in non-Hodgkin Lymphoma    with Reshaped Human Monoclonal Antibody CAMPATH-1H”, Lancet ii    1394-1398.-   [24] Harlan, J. M, 1985, Blood 65: 513-526, “Leukocyte-endothelial    interactions”-   [25] Collins, et al., 1986, Proc. Natl. Acad. Sci. USA 83: 446-450,    “Recombinant Human Tumor Necrosis Factor Increases mRNA Levels and    Surface Expression of HLA-A, B antigens in vascular endothelial    cells and dermal fibroblasts in vitro”-   [26] Pober et al., 1986, “Overlapping Pattern of Activation of Human    Endothelial Cells by Interleukin-1, Tumor Necrosis Factor, and    Immune Interferon, J. Immunol. 137: 1893-1896-   [27] Bevilacqua, et al., 1987, Proc. Natl. Acad. Sci. USA 84:    9238-9242, “Identification of an Inducible Endothelial-Leukocyte    Adhesion Molecule”-   [28] Leeuwenberg, et al., 1989, “Induction of an Activation Antigen    on Human Endothelial Cells in vitro, Eur. J. Immunol. 19: 715-729-   [29] Bevilacqua, et al., 1989, “Endothelial leukocyte adhesion    molecule 1; an inducible receptor for neutrophils related to    complement regulatory proteins and lectins, Science 243:1160-1165.-   [30] Dustin, et al., 1986, “Induction by IL-1 and Interferon-γ:    tissue distribution, biochemistry, and function of a natural    adherence molecule (ICAM-1), J. Immunol. 137:245-254-   [31] Boyd et al., 1988, “Intercellular adhesion molecule 1 (ICAM-1)    has a central role in cell-cell contact-mediated immune mechanisms,    Proc. Natl. Acad. Sci. USA 85: 3095-3099-   [32] Dustin and Springer, 1988, “Lymphocyte function-associated    antigen-1 (LFA-1) Interaction with Intercellular Adhesion Molecule-1    (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion    to cultured endothelial cells”, J. Cell Biol. 107:321-331-   [33] Osborn et al., 1989, “Direct Cloning of Vascular Cell Adhesion    Molecule 1, a cytokine-induced endothelial protein that binds to    lymphocytes, Cell 59: 1203-1211-   [34] Hynes, 1987, “Integrins: a family of cell surface receptors”    Cell 48: 549-554-   [35] Marcantonio and Hynes, 1988, “Antibodies to the conserved    cytoplasmic domain of the integrin β₁ subunit react with proteins in    vertebrates, invertebrates and fungi, J. Cell Biol. 106: 1765-1772-   [36] Kishimoto et al., 1989, “The leukocyte integrins”, Adv.    Immunol. 46: 149-182-   [37] Ruoslahti, 1988, “Fibronectin and its receptors” Annu. Rev.    Biochem. 57: 375-413-   [38] Hemler et al., 1990, “VLA proteins in the integrin family:    structures, functions and their role on leukocytes” Annu Rev.    Immunol. 8:365-400-   [39] Hemler et al., 1987, “Characterization of the cell surface    heterodimer VLA-4 and related peptides” J. Biol. Chem.    262:11478-11485-   [40] Clayberger, et al., 1987, “Identification and Characterization    of two novel lymphocyte function-associated antigens, L24 and    L25” J. Immunol. 138:1510-1514-   [41] Takada et al., 1989, “The Primary Structure of the α4 subunit    of VLA-4; homology to other integrins and a possible cell-cell    adhesion function:, EMBO J. 8:1361-1368-   [42] Holtzmann et al., 1989, “Identification of a murine Peyer's    patch-specific lymphocyte homing receptor as an integrin molecule    with an α chain homologous to human VLA-4α,” Cell 56:37-46-   [43] Bednarczyk and McIntyre, 1990, “A monoclonal antibody to    VLA-4-α chain (CDw49) induces homotypic lymphocyte aggregation”, J.    Immunol. in press-   [44] Wayner et al., 1989, “Identification and characterization of    the lymphocyte adhesion receptor for an alternative cell attachment    domain in plasma fibronectin”, J. Cell Biol. 109:1321-1330-   [45] Elices et al., 1990, “VCAM-1 on Activated Endothelium Interacts    with the Leukocyte Integrin VLA-4 at a Site Distinct from the    VLA-4/Fibronectin Binding Site”, Cell 60:577-584-   [46] Rice et al., 1989, “An inducible endothelial cell surface    glycoprotein mediates melanoma adhesion,” Science, 246:1303-1306-   [47] Cybulsky, M. I. and Gimbrone, M. A., Jr. 1991, “Endothelial    expression of a mononuclear leukocyte adhesion molecule during    atherogenesis,” Science, 251:788-791-   [48] Freedman et al., 1990, “Adhesion of human B cells to germinal    centers in vitro involves VLA-4 and INCAM-110,” Science,    249:1030-1033-   [49] Miyake et al., 1991, “A VCAM-like adhesion molecule on murine    bone marrow stromal cells mediates binding of lymphocyte precursors    in culture,” J. Cell Biol., 114:557-565-   [50] Polte et al., 1990, “Full length vascular cell adhesion    molecule 1 (VCAM-1),” Nuc. Ac. Res., 18:5901-   [51] Hession et al., 1991, “Cloning of an alternate form of vascular    cell adhesion molecule-1 (VCAM1)”, J. Biol. Chem., 266:6682-6685-   [52] Osborn and Benjamin, U.S. Ser. No. 07/821,712 filed Sep. 30,    1991-   [53] Carlos et al., 1990, “Vascular cell adhesion molecule-1    mediates lymphocyte adherence to cytokine-activated cultured human    endothelial cells,” Blood, 76, 965-970-   [54] Pulido et al., 1991, “Functional Evidence for Three Distinct    and Independently Inhibitable Adhesion Activities Mediated by the    Human Integrin VLA-4,” J. Biol. Chem., 266(16):10241-10245-   [55] Sanchez-Madrid et al., 1986, “VLA-3: A novel polypeptide    association within the VLA molecular complex: cell distribution and    biochemical characterization,” Eur. J. Immunol., 16:1343-1349-   [56] Weller et al., 1991, “Human eosinophil adherence to vascular    endothelium mediated by binding to vascular cell adhesion molecule 1    and endothelial leukocyte adhesion molecule 1,” Proc. Natl. Acad.    Sci. USA, 4488:7430-7433-   [57] Walsh et al., 1991, “Human Eosinophil, But Not Neutrophil,    Adherence to IL-1-Stimulated Human Umbilical Vascular Endothelial    Cells Is α₄β₁ (Very Late Antigen-4) Dependent,” J. Immunol.,    146:3419-3423-   [58] Bochner et al., 1991, “Adhesion of Human Basophils,    Eosinophils, and Neutrophils to Interleukin 1-activated Human    Vascular Endothelial Cells: Contributions of Endothelial Cell    Adhesion Molecules,” J. Exp. Med., 173:1553-1556-   [59] Dobrina et al., 1991, “Mechanisms of Eosinophil Adherence to    Cultured Vascular Endothelial Cells,” J. Clin. Invest., 88:20-26-   [60] Lobb, U.S. Ser. No. 07/821,768 filed Jan. 13, 1992-   [61] Lobb, U.S. Ser. No. 07/835,139 filed Feb. 12, 1992-   [62] Papayannopoulou, U.S. Ser. No. 07/977,702 filed Nov. 13, 1992-   [63] Favoloro et al., 1980, “Transcriptional Maps of Polyome Virus    Specific RNA: Analysis by Two-Dimensional Nuclease S1 Gel Mapping”,    Methods in Enzymology 65:718-749.-   [64] Orlandi et al., 1989, “Cloning immunoglobulin variable domains    for expression by the polymerase chain reaction”, Proc. Natl. Acad.    Sci. USA 86:3833-3837.-   [65] Huse et al., 1989, “Generation of a Large Combinational Library    of Immunoglobulin Repertoire in Phage Lambda”, Science    246:1275-1281.-   [66] Jones and Bendig, 1991, “Rapid PCR-Cloning of Full-length Mouse    Immunoglobulin Variable Regions”, Biotechnology 9:88-89-   [67] Saiki et al., 1988, “Primer-Directed Enzymatic Amplification of    DNA with a Thermostable DNA Polymerase”, Science 239:487-491-   [68] Molecular Cloning. A Laboratory Manual, 1982, eds. T. Maniatis    et al., published by Cold Spring Harbor Laboratory, Cold Spring    Harbor, N.Y.-   [69] Sanger et al., 1977, “DNA Sequencing with Chain-terminating    Inhibitors”, Proc. Natl. Acad. Sci. USA 74:5463-5467.-   [70] Takahashi et al., 1982, “Structure of Human Immunoglobulin    Gamma Genes: Implications for Evolution of a Gene Family”, Cell    29:671-679,-   [71] Flanagan and Rabbitts, 1982, “Arrangement of Human    Immunoglobulin Heavy Chain Construct Region Genes Implies    Evolutionary Amplification of a Segment Containing γ, ε and α    genes”, Nature 300:709-713.-   [72] Hieter, 1980, “Cloned Human and Mouse Kappa Immunoglobulin    Constant and J. Region Genes Conserve Homology in Functional    Segments”, Cell 22:197-207-   [73] Kilmartin et al., 1982, “Rat Monoclonal Antitubulin Antibodies    Derived by Using a New Non-secreting Rat Cell Line”, J. Cell Biol.    93:576-582.-   [74] Ho et al., 1989, “Site-directed Mutagenesis by Overlap    Extension Using The Polymerase Chain Reaction”, Gene 77:51-59-   [75] Burkly et al., U.S. Ser. No. 07/916,098, filed Jul. 24, 1992-   [76] Bebbington et al., 1992, “High-Level Expression of a    Recombinant Antibody from Myeloma Cells Using A Glutamine Synthetase    Gene as an Amplifiable Selectable Marker”, Bio/Technology    10:169-175.-   [77] WO86/05807 (Celltech Limited)-   [78] WO87/04462 (Celltech Limited)-   [79] WO89/01036 (Celltech Limited)-   [80] WO89/10404 (Celltech Limited)-   [81] Lobb et al., 1991a, “Expression and Functional Characterization    of a Soluble Form of Vascular Cell Adhesion Molecule 1”, Biochem.    Biophys. Res. Comm. 178:1498-1504-   [82] Lobb et al., 1991b, “Expression and Functional Characterization    of a Soluble Form of Endothelial-Leukocyte Adhesion Molecule 1”, J.    Immunol. 147:124-129

Each of the above-listed references is hereby incorporated by referencein its entirety.

1-45. (canceled)
 46. A humanized recombinant antibody molecule, or anα4-binding fragment thereof, comprising: at least one antibody heavychain, or an α4-binding fragment thereof, comprising non-human CDRs atpositions 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) (Kabat numbering)from a mouse anti-α4 antibody and framework regions from a humanizedheavy chain variable region based on the HP1/2 heavy chain variableregion (SEQ ID NO: 6); and at least one antibody light chain, or anα4-binding fragment thereof, comprising non-human CDRs at positions24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) from a mouse anti-α4antibody and framework regions from a humanized light chain variableregion based on the HP1/2 light chain variable region (SEQ ID NO: 10),wherein said HP1/2 framework regions have non-human residues atframework positions 60 and 67.